Level Of Transcription Initiation Research Articles

The SMN genes are subject to transcriptional regulation during cellular differentiation

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of α-motor neurons and muscular atrophy. The causal survival motor neuron ( SMN) gene maps to a complex region of chromosome 5q13 harbouring an inverted duplication. Thus, there are two SMN genes, SMN1 and SMN2, but SMN1-deficiency alone causes SMA. In this study we demonstrate, for the first time, down-regulation of SMN promoter activity during cellular differentiation. Specifically, the minimal SMN promoter is four times more active in undifferentiated embryonal carcinoma P19 cells compared to cells treated with retinoic acid (RA) to initiate neuronal differentiation. This effect is mediated by sequences contained within the minimal core promoter that we have confined to the 257 nucleotides upstream of exon 1. We have identified seven regions that are highly conserved between the mouse and human SMN core promoters and this region contains the consensus sequence for a number of transcription factors. Most notably, AhR, HNF-3 and N-Oct3 have already been shown to respond to RA treatment of EC cells, while E47, HNF-3, MAZ, N-Oct3 and Pit-1a have been implicated in embryonic, muscle or neural development. In addition, we have mapped two strong transcription initiation sites upstream of SMN exon 1. The novel −79 site identified in this study is preferentially utilized during human foetal development. Furthermore, analysis of RNA from SMA patients with deletions of the entire SMN1 gene or chimpanzees that lack SMN2 suggests that the level of transcription initiation at these sites may be different for the SMN1 and SMN2 genes. Taken together, this work provides the first demonstration of transcriptional regulation of these genes during cellular differentiation and development. Deciphering the underlying mechanisms responsible for regulating SMN transcription may provide important clues towards enhancing SMN2 gene expression, one target for the treatment of SMA.

Expression of the Bacillus subtilis sulphonate–sulphur utilization genes is regulated at the levels of transcription initiation and termination

The Bacillus subtilis ssuBACD gene cluster is required for the utilization of aliphatic sulphonates as sulphur sources. The regulation of expression of the ssu genes was studied in constructs carrying chromosomal transcriptional fusions of the ssu promoter region to lacZ. When sulphate or cystine served as sulphur sources, expression of the ssu genes was repressed. A putative terminator located between the promoter and the start of the ssuB gene partially overlaps a putative antiterminator. Removal of both the antiterminator and the terminator led to a decrease in the repression ratio of about 10-fold, but not to constitutive expression. Replacement of the ssu promoter by the Pspac promoter led to decreased expression of the ssu genes, but not to loss of repression by sulphate and cystine. Thus, the repression exerted by sulphate and cystine resulted from regulation at the level of both transcription initiation and transcription termination. O-acetyl-L-serine, a precursor of cysteine, served as effector molecule in both regulation systems.

Expression of the Bacillus subtilis sulphonate-sulphur utilization genes is regulated at the levels of transcription initiation and termination

The Bacillus subtilis ssuBACD gene cluster is required for the utilization of aliphatic sulphonates as sulphur sources. The regulation of expression of the ssu genes was studied in constructs carrying chromosomal transcriptional fusions of the ssu promoter region to lacZ. When sulphate or cystine served as sulphur sources, expression of the ssu genes was repressed. A putative terminator located between the promoter and the start of the ssuB gene partially overlaps a putative antiterminator. Removal of both the antiterminator and the terminator led to a decrease in the repression ratio of about 10-fold, but not to constitutive expression. Replacement of the ssu promoter by the Pspac promoter led to decreased expression of the ssu genes, but not to loss of repression by sulphate and cystine. Thus, the repression exerted by sulphate and cystine resulted from regulation at the level of both transcription initiation and transcription termination. O-acetyl-l-serine, a precursor of cysteine, served as effector molecule in both regulation systems.

Multiple modes of transcriptional regulation by the HIV-1 Tat transactivator.

Regulation of HIV-1 gene expression by the viral Tat transactivator is a critical step in the viral life cycle. Tat acts as a highly unusual transcription factor that interacts with a stem-loop RNA structure (TAR) found at the 5' end of all viral transcripts. There, it induces a modification of chromatin at the HIV-1 long terminal repeat (LTR) promoter and stimulates the recruitment of elongation-competent RNA polymerase II complexes capable of processive transcription. Increase of transcriptional elongation is the consequence of the interaction of Tat with cyclin T1, the cyclin component of CDK9, which phosphorylates the carboxy-terminal domain of RNA polymerase II to enhance its processivity. Tat-induced transcriptional activation of the LTR promoter is concomitant with recruitment of the transcriptional coactivators p300 and the highly homologue cAMP-responsive transcription factor binding protein (CBP). These large proteins act at the level of transcriptional initiation by bridging the basal transcription machinery with specific transcriptional activators. Furthermore, p300/CBP are histone acetyl-transferases capable of modulating the interaction of nucleosomes with DNA and with chromatin remodeling complexes. Besides histones, Tat itself is a substrate for the enzymatic activity of p300/CBP and of the associated factor P/CAF, suggesting a regulatory role of acetylation on the protein itself. Devising a unifying model for LTR activation that includes activities of Tat at the levels of both transcriptional initiation and transcriptional elongation is a challenging task at this moment. Nevertheless, protein localization studies indicate that both cyclin T1 and p300/CBP co-localize in specific subnuclear compartments, thus suggesting participation of both proteins in the formation of multimolecular complexes governing coordinated steps of transcriptional activation.

Functional Disruption of the CD28 Gene Transcriptional Initiator in Senescent T Cells

We recently reported that aging is accompanied by the emergence of CD4(+)CD28(null) T cells, a functionally aberrant lymphocyte subset rarely seen in individuals younger than 40 years. Here, we directly examined whether the lack of CD28 expression is due to a defect at the level of transcriptional initiation. Molecular studies reveal that CD28 gene transcription is controlled by two sequence motifs, sites alpha and beta. In vitro transcription assays using initiator-dependent DNA templates revealed that reversed polarity or the deletion of either motif inhibited transcription, indicating that alpha/beta sequences constitute a composite initiator. Moreover, nuclear extracts from CD28(null) cells failed to activate transcription of alphabeta-initiator DNA templates. Transcription of such templates was, however, restored with the addition of extracts from CD28(+) cells. Although previously described initiator elements have been defined by a consensus sequence, the alphabeta-initiator has no homology to such sequence. These studies demonstrate that initiators have functions other than positioning elements for the basal transcription complex. Rather, initiators can have a direct role in regulating the expression of specific genes. The gain or loss of initiator activity can be an important determinant of cell phenotypes.

Transcriptional control of the sulfur-regulated cysH operon, containing genes involved in L-cysteine biosynthesis in Bacillus subtilis.

The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in Bacillus subtilis and other gram-positive bacteria. In this study we describe the expression pattern of the B. subtilis cysH operon in response to sulfur starvation. A 6.1-kb polycistronic transcript which includes the cysH, cysP, ylnB, ylnC, ylnD, ylnE, and ylnF genes was identified. Its synthesis was induced by sulfur limitation and strongly repressed by cysteine. The cysH operon contains a 5' leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system. Here we show that induction of B. subtilis cysH operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription. Deletion of a 46-bp region adjacent to the -35 region of the cysH promoter led to high-level expression of the operon, even in the presence of cysteine. We also found that O-acetyl-L-serine (OAS), a direct precursor of cysteine, renders cysH transcription independent of sulfur starvation and insensitive to cysteine repression. We propose that transcription of the cysH operon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS. Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of cysH expression. These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.

Glucocorticoid mediated transcriptional repression of c- myc in apoptotic human leukemic CEM cells

Suppression of c- myc has been implicated as a critical event in some glucocorticoid-evoked apoptotic systems. It is therefore of interest to understand the mechanism of glucocorticoid-regulation of the c- myc gene. In the present study, a detailed analysis of dexamethasone (Dex)-evoked regulation of the human c-myc gene in human leukemic CEM-C7 cells has been performed. Dex suppresses c- myc mRNA and immunoreactive protein expression in clone CEM-C7 and subclone CEM-C7-14 cells. Nuclear run-on assays suggested that the regulation occurred at the level of transcription initiation. The half-life of c- myc mRNA was approximately 30 min and its stability was not affected by Dex treatment. In addition, Dex suppressed luciferase gene expression driven by −2052 to +34 bp c- myc promoter in transfected CEM-C7-14 cells. This result further supports that c- myc gene is suppressed by Dex at the transcriptional level in apoptotic human leukemic cells.

Regulation of c-myc Transcription by Interleukin-2 (IL-2): IDENTIFICATION OF A NOVEL IL-2 RESPONSE ELEMENT INTERACTING WITH STAT-4

Regulation of c-myc expression is known to occur at the level of transcription initiation. However, the participating promoter elements and their cognate binding proteins have not been fully characterized. c-myc transcription can be stimulated by a number of cytokines including interleukin-2 (IL-2). We have identified a novel IL-2-responsive element, located in the 5'-flanking region of the c-myc gene, between nucleotides -1406 and -1387 (relative to the P2 promoter). This element belongs to the family of interferon-gamma activation site-like responsive elements and has the core sequence TTCCAATAA. We confirmed that IL-2-mediated signaling involves activation by phosphorylation of Jak2 tyrosine kinase and subsequently STAT4. The transcription factor STAT4 binds the TTCCAATAA motif within this responsive element and, therefore, is probably involved in enhancing c-myc transcription upon IL-2 stimulation. Our results propose participation of Jak2 and STAT4 in IL-2-induced up-regulation of c-myc.

Regulation of the Wilms' tumour suppressor protein transcriptional activation domain.

The Wilms' tumour suppressor protein WT1 contains a transcriptional regulatory domain that can either activate or repress transcription depending upon its cellular environment. The mechanistic basis for this dichotomy is unclear however. Here, we dissect the transcriptional regulatory domains of WT1. We find that a region within the domain of WT1 attributed to transcriptional repression is a potent suppressor of the activation domain at several promoters and in different cell types. In vitro transcription analysis suggests that the mechanism of suppression of the activation domain occurs at the level of transcription initiation. Furthermore we find that the WT1 suppression domain is able to inhibit a heterologous activation domain when fused in cis. Dissection of this domain resulted in the delineation of a 30 amino acid region that was sufficient to confer suppression of a transcriptional activation domain both in vivo and in vitro. Additionally, we find that the WT1 transcriptional activation domain interacts with the general transcription factor TFIIB and that this interaction is not affected by the suppression domain. Taken together, these studies suggest that the suppression domain of WT1 interacts with a cosuppressor protein to mediate inhibition of the WT1 transcriptional activation domain.

Cloning and functional analysis of the human IRF-3 promoter.

We have isolated a genomic clone of the human IRF-3 gene containing 779 nucleotides of the 5' flanking region and the complete intron exon sequence. The gene has eight exons which span about 6 kb on chromosome 19q13.3. The IRF-3 promoter has neither a conserved TATA box nor a CCAAT box motif but is GC rich. Several putative DNA-binding elements were identified, including three SP-1 sites, a USF element, a HOX box, a CarG box, and an NF-1 site. Deletion analysis of the promoter region showed that the core basal promoter, consisting of 113 bp 5' of the first transcription start site, was sufficient for constitutive expression. This region contains only one of the SP-1 sites as well as the HOX element and NF-1 site, and although it is GC rich, it does not contain any of the other putative DNA-binding sites. In contrast, the mouse IRF-3 promoter, while displaying a high degree of homology with the human promoter, contains both TATA and CCAAT box motifs, suggesting that, at least at the level of transcription initiation, these genes may be differentially regulated.

Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei.

Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10(-7) per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated.

Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif.

Insects possess a powerful immune system, which in response to infection leads to a vast production of different antimicrobial peptides. The regulatory regions of many immunity genes contain a GATA motif in proximity to a kappaB motif. Upon infection, Rel proteins enter the nucleus and activate transcription of the immunity genes. High levels of Rel protein-mediated Cecropin A1 expression previously have been shown to require the GATA site along with the kappaB site. We provide evidence demonstrating that the GATA motif is needed for expression of the Cecropin A1 gene in larval fat body, but is dispensable in adult fat body. A nuclear DNA-binding activity interacts with the Cecropin A1 GATA motif with the same properties as the Drosophila GATA factor Serpent. The GATA-binding activity is recognized by Serpent-specific antibodies, demonstrating their identity. We show that Serpent is nuclear in larval fat body cells and haemocytes both before and after infection. After overexpression, Serpent increases Cecropin A1 transcription in a GATA-dependent manner. We propose that Serpent plays a key role in tissue-specific expression of immunity genes, by priming them for inducible activation by Rel proteins in response to infection.

Introns are necessary for mRNA accumulation in Schizophyllum commune.

The cDNA coding sequence of the Agaricus bisporus hydrophobin gene ABH1 under the regulation sequences of the Schizophyllum commune SC3 hydrophobin gene gave no expression in S. commune. In contrast, the genomic coding sequence (containing three introns) produced high levels of ABH1 mRNA when transformed to S. commune in the same configuration. Apparently, introns were needed for the accumulation of mRNAs from the ABH1 gene. When the effect of intron deletion on expression of the homologous genes SC3 and SC6 was examined, it was observed that only the genomic coding sequences were expressed in S. commune. Run-on analysis with nuclei harbouring intron-containing and intronless SC6 showed that this effect did not occur at the level of transcription initiation: genomic and cDNA sequences were equally active in this respect. When a 50 bp artificial intron containing the consensus splice and branch sites of S. commune introns, in addition to random-generated sequences, was introduced in the right orientation into the intronless SC3 transcriptional unit, accumulation of SC3 mRNA was restored. By polymerase chain reaction amplification, no unspliced SC3 mRNA species could be detected. Furthermore, the addition of an intron into the transcriptional unit of the gene for green fluorescent protein (GFP) effected clear fluorescence of the transgenic hyphae. Apparently, splicing is required for the normal processing of primary transcripts in S. commune.

Stimulation of Tat-associated kinase-independent transcriptional elongation from the human immunodeficiency virus type-1 long terminal repeat by a cellular enhancer.

The human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) initiates transcription efficiently but produces only short transcripts in the absence of the trans-activator protein, Tat. To determine whether a cellular enhancer could provide the signals required to recruit an elongation-competent polymerase to the HIV-1 LTR, the B cell-specific immunoglobulin heavy chain gene enhancer (IgHE) was inserted upstream of the LTR. The enhancer increased transcription in the absence of Tat between 6- and 7-fold in transfected B cells, but the full-length transcripts remained at basal levels in HeLa cells, where the enhancer is inactive. RNase-protection studies showed that initiation levels in the presence and absence of the enhancer were constant, but the enhancer significantly increased the elongation capacity of the polymerases. Tat-stimulated elongation is strongly inhibited by the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which inhibits the Tat-associated kinase, TAK (CDK9). However, polymerases initiating transcription from LTRs carrying the enhancer were able to efficiently elongate in the presence of DRB. Specific repression of TAK by expression in trans of the CDK9 kinase also inhibited Tat-stimulated elongation but did not inhibit enhancer-dependent transcription significantly. Thus, the activation of polymerase processivity by the IgHE involves a unique mechanism which is independent of TAK.

Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum.

Members of the Plasmodium falciparum var gene family encode clonally variant adhesins, which play an important role in the pathogenicity of tropical malaria. Here we employ a selective panning protocol to generate isogenic P.falciparum populations with defined adhesive phenotypes for CD36, ICAM-1 and CSA, expressing single and distinct var gene variants. This technique has established the framework for examining var gene expression, its regulation and switching. It was found that var gene switching occurs in situ. Ubiquitous transcription of all var gene variants appears to occur in early ring stages. However, var gene expression is tightly regulated in trophozoites and is exerted through a silencing mechanism. Transcriptional control is mutually exclusive in parasites that express defined adhesive phenotypes. In situ var gene switching is apparently mediated at the level of transcriptional initiation, as demonstrated by nuclear run-on analyses. Our results suggest that an epigenetic mechanism(s) is involved in var gene regulation.

Transcriptional regulation of FABP expression in flight muscle of the desert locust, Schistocerca gregaria

FABP is the most abundant cytosolic protein in developed flight muscle of adult locusts, but it is completely absent in nymphs. During the two weeks following adult ecdysis, FABP rises to 18% of the total soluble proteins. Its mRNA increases steadily up to day 8, before it gradually declines until reaching a low concentration at day 15, which remains constant for the rest of the animal's life. Using a PCR primer combination specific for a 597 bp sequence of intron 1, we have developed a reverse transcription PCR assay to quantify the amount of primary transcript present in muscle tissue at various ages. The FABP gene is not transcribed prior to metamorphosis; its primary transcript rises rapidly during the first two days of adult life, and remains close to maximal until day 5. Subsequently its level rapidly declines, ultimately reaching values of less than 0.02% of the maximal level. The correlation between primary transcript, mRNA and FABP content were analyzed by modeling transcription, translation and degradation with computer modeling software. The computer simulation is in good agreement with the experimentally obtained data, suggesting that the control of FABP expression in locust flight muscle occurs predominantly at the level of transcription initiation.

Male-specific transcription initiation of the C4-Slp gene in mouse liver follows activation of STAT5.

The mouse genes encoding the constitutively expressed complement component C4 and its closely related isoform C4-Slp (sex-limited protein), which is expressed only in male animals of several strains, provide a unique model to study sequence elements and trans-acting factors responsible for androgen responsiveness. Our previous studies have shown that hormonal induction of C4-Slp is mediated by a sex-specific pattern of growth hormone secretion. Promoter analyses in vitro have led to contradictory conclusions concerning the significance of C4-Slp-specific sequences in the 5' flanking region. Mutant mice carrying the H-2(aw18) haplotype, which is characterized by a large deletion in the S region covering the C4 and 21-OHase A genes, permit the direct in vivo analysis of C4-Slp transcription, unhindered by the presence of C4. Run-on analysis of transcription in liver nuclei of males and females of this strain demonstrated a 100-fold higher transcriptional activity in males, essentially determined at the transcription initiation level. The androgen dependence of transcription initiation was confirmed by run-on analysis of testosterone-treated females, where transcriptional activity started after 6 days of androgen treatment and reached male levels after 20 days. Conversely, the growth hormone-regulated activity of transcription factor STAT5 was already detected in liver nuclei after 48 hr of androgen treatment. Furthermore, we demonstrate that activated STAT5 recognizes in vitro two upstream gamma interferon-activated sequence (GAS) elements of the C4-Slp gene, centered at positions -1969 and -1831. We postulate that binding of STAT5 to these C4-Slp-specific GAS elements plays a crucial role in the chromatin remodelings that lead to transcriptional competence of the C4-Slp gene in the liver.

Selective down-regulation of human papillomavirus transcription by 2-deoxyglucose.

The glycolytic pathway inhibitor 2-deoxyglucose (2-DG) is capable of suppressing the transcription of the human pathogenic papillomavirus type 18 (HPV 18) in cervical carcinoma cells and derived non-tumorigenic somatic cell hybrids at the level of transcription initiation. HPV down-regulation is selective, since other reference genes are not affected or even up-regulated under the same experimental conditions. Moreover, 2-DG appears to restore the normal half-life of the tumor suppressor gene product p53, because the protein is strongly up-regulated after HPV 18 E6/E7 suppression. The observed 2-DG-effect is not cytotoxic and is reversible after refeeding with fresh medium. HPV 18 suppression by 2-DG can be completely abrogated by simultaneous treatment with the intracellular Ca2+ antagonist TMB-8, indicating that Ca2+, a known intracellular "second messenger", is involved in this process. Elevated c-myc and p53 expression appears to be responsible for the time-dependent accumulation of apoptotic cells after prolonged 2-DG treatment. The finding that 2-DG acts selectively against the expression of a human pathogenic papillomavirus strongly suggests that an appropriate level of glycolysis is not only a peculiarity of growing tumors, but even may be an essential prerequisite for the maintenance of virus-specific E6/E7 gene expression. Our results may have substantial implications for the potential therapeutic application of 2-DG or other glucose derivatives in the treatment of precancerous and malignant HPV-associated lesions.

Control of Adenovirus Early Gene Expression during the Late Phase of Infection

The adenovirus gene regulatory program occurs in two distinct phases, as defined by the onset of DNA replication. During the early phase, the E1A, E1B, E2, E3, and E4 genes are maximally expressed, while the major late promoter (MLP) is minimally expressed and transcription is attenuated. After the onset of DNA replication, the IVa2 and pIX genes are expressed at high levels, transcription from the MLP is unattenuated and fully activated, and early gene expression is repressed. Although the cis elements and trans-acting factors responsible for the late-phase activation of the MLP have been identified and characterized and the role of DNA replication in activation has been established, the mechanism(s) underlying the commensurate decrease in early gene expression has yet to be elucidated. The results of this study demonstrate that this decrease depends on a fully functional MLP. Specifically, virus mutants with severely deficient transcription from the MLP exhibit a marked increase in expression of the E1A, E1B, and E2 early genes. These increases were observed at the level of transcription initiation, mRNA accumulation, and protein production. In addition, expression from the late gene pIX, which is not contained within the major late transcription unit (MLTU), is also markedly increased. To begin the analysis of the mechanisms underlying these late-phase effects, mixed-infection experiments with mutant and wild-type viruses were performed. The results show that the effects on early gene expression, as measured both at the protein and RNA levels, are mediated in trans and not in cis. These observations are consistent either with a model in which one or more late protein products encoded by the MLTU acts as a repressor of early gene expression or with one in which the wild-type MLP competes with early promoters for limiting transcription factors.

Interleukin-5 mRNA stability in human T cells is regulated differently than interleukin-2, interleukin-3, interleukin-4, granulocyte/macrophage colony-stimulating factor, and interferon-gamma.

Interleukin-5 (IL-5) transcriptional activation and mRNA stability were investigated in a human TH0 T-cell clone (SP-B21) and in nonclonal CD4 TH2 cells, differentiated in vitro from peripheral blood T cells. Cells were stimulated with alpha-CD3 monoclonal antibody (mAb) with and without alpha-CD28 mAb. Comparison to other cytokine genes revealed aspects of mRNA regulation unique to IL-5. The half-life (t1/2) of IL-5 mRNA, determined by addition of actinomycin D (ActinoD) or cyclosporin A (CSA) was longer (by >= 2 h) than that of IL-2, IL-3, IL-4, interferon-gamma, or granulocyte/macrophage colony-stimulating factor. With the exception of IL-5, t1/2 values were significantly shorter with CSA as the transcriptional inhibitor than with ActinoD. The t1/2 value of IL-5 mRNA, but not the other cytokine transcripts, determined with either ActinoD or CSA, was longer than predicted from the kinetics of steady-state mRNA decline. Co-stimulation of both cell types with alpha-CD28 mAb increased the stability of cytokine transcripts weakly, and IL-5 remained the most stable transcript. Thus, the degradation pathway that targets IL-5 is distinct from the other cytokine transcripts measured and involves proteins whose transcription is blocked by ActinoD and CSA. From examination of the levels of transcription initiation (nuclear run-on assay) and steady-state mRNA attained in cultures stimulated in the presence of the protein synthesis inhibitor, cycloheximide, only IL-5 transcription initiation had an absolute dependency on new protein synthesis.