Core Promoter Sequences Research Articles

A functionally distinct TATA box required for late progression through the Epstein-Barr virus life cycle.

During EBV infection, lytic DNA replication activates late gene expression in trans via an uncharacterized pathway. In this study, we mapped the target of this regulatory cascade to a variant TATA box (TATTAAA) and the 3' flanking region within the core promoter of the BcLF1 gene. The inherent late activity of this core promoter is, surprisingly, disrupted by a heterologous enhancer, suggesting that late gene expression is regulated through core promoter sequences located in a transcriptionally inert environment.

The Novel Core Promoter Element GAAC in the hgl5 Gene of Entamoeba histolytica Is Able to Direct a Transcription Start Site Independent of TATA or Initiator Regions

Entamoeba histolytica, an enteric protozoa, is the third leading parasitic cause of death worldwide. Investigation of the transcriptional machinery of this eukaryotic pathogen has revealed an unusual core promoter structure that consists of nonconsensus TATA and initiator regions and a novel third conserved core promoter sequence, the GAAC element. Mutation of this region in the hgl5 promoter decreases reporter gene expression and alters the transcription start site. Using positional analysis of this element, we have now demonstrated that it is able to direct a new transcription start site, 2-7 bases downstream of itself, independent of TATA and Inr regions. The GAAC region was also shown to control the rate of transcription via nuclear run on analysis and an amebic nuclear protein was demonstrated to specifically interact with this sequence. This is the first description in the eukaryotic literature of a third conserved core promoter element, distinct from TATA or initiator regions, that is able to direct a transcription start site. We have formulated two models for the role of the GAAC region: (i) the GAAC-binding protein is a part of the TFIID complex and (ii) the GAAC-binding protein functions to "tether" TATA-binding protein to the TATA box.

Budesonide epimer R or dexamethasone selectively inhibit platelet-activating factor-induced or interleukin 1beta-induced DNA binding activity of cis-acting transcription factors and cyclooxygenase-2 gene expression in human epidermal keratinocytes.

To further understand the molecular mechanism of glucocorticoid action on gene expression, DNA-binding activities of the cis-acting transcription factors activator protein 1 (AP1), AP2, Egr1 (zif268), NF-kappaB, the signal transducers and activators of transcription proteins gamma interferon activation site (GAS), Sis-inducible element, and the TATA binding protein transcription factor II D (TFIID) were examined in human epidermal keratinocytes. The cytokine interleukin 1beta (IL-1beta) and platelet-activating factor (PAF), both potent mediators of inflammation, were used as triggers for gene expression. Budesonide epimer R (BUDeR) and dexamethasone (DEX) were studied as potential antagonists. BUDeR or DEX before IL-1beta- or PAF-mediated gene induction elicited strong inhibition of AP1-, GAS-, and in particular NF-kappaB-DNA binding (P < 0.001, ANOVA). Only small effects were noted on AP2, Egr1 (zif268), and Sis-inducible element-DNA binding (P > 0.05). No significant effect was noted on the basal transcription factor TFIID recognition of TATA-containing core promoter sequences (P > 0.68). To test the hypothesis that changing cis-acting transcription factor binding activity may be involved in inflammatory-response related gene transcription, RNA message abundance for human cyclooxygenase (COX)-1 and -2 (E.C.1.14.99.1) was assessed in parallel by using reverse transcription-PCR. Although the COX-1 gene was found to be expressed at constitutively low levels, the TATA-containing COX-2 gene, which contains AP1-like, GAS, and NF-kappaB DNA-binding sites in its immediate promoter, was found to be strongly induced by IL-1beta or PAF (P < 0.001). BUDeR and DEX both suppressed COX-2 RNA message generation; however, no correlation was associated with TFIID-DNA binding. These results suggest that on stimulation by mediators of inflammation, although the basal transcription machinery remains intact, modulation of cis-activating transcription factor AP1, GAS, and NF-kappaB-DNA binding by the glucocorticoids BUDeR and DEX play important regulatory roles in the extent of specific promoter activation and hence the expression of key genes involved in the inflammatory response.

Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro.

RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inflorescence that supports promoter-dependent RNA pol I transcription in vitro. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain approximately 30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.

The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo

The Drosophila gypsy retrotransposon disrupts gene activity by blocking the interactions of distal enhancers with target promoters. This enhancer-blocking activity is mediated by a 340 bp insulator DNA within gypsy. The insulator contains a cluster of binding sites for a zinc finger protein, suppressor of Hairy wing [su(Hw)]. Recent studies have shown that a second protein, mod(mdg4), is also important for normal insulator function. Mutations in mod(mdg4) exert paradoxical effects on different gypsy-induced phenotypes. For example, it enhances yellow2 but suppresses cut6. Here, we employ a stripe expression assay in transgenic embryos to investigate the role of mod(mdg4) in gypsy insulator activity. The insulator was inserted between defined enhancers and placed among divergently transcribed reporter genes (white and lacZ) containing distinct core promoter sequences. These assays indicate that mod(mdg4) is essential for the enhancer-blocking activity of the insulator DNA. Moreover, reductions in mod(mdg4)+ activity cause the insulator to function as a promoter-specific silencer that selectively represses white, but not lacZ. The repression of white does not affect the expression of the closely linked lacZ gene, suggesting that the insulator does not propagate changes in chromatin structure. These results provide an explanation for why mod(mdg4) exerts differential effects on different gypsy-induced mutations.

The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription.

The lactose (lac) operon promoter is positively regulated by the catabolite gene activator-cyclic AMP complex (CAP) that binds to the DNA located 61.5 bp upstream of the transcription start site. Between the CAP binding site and the core promoter sequence is a 13-bp sequence (from -38 to -50 [the -45 region]). The possible roles of the -45 region in determining the CAP-independent level of lac expression and in the CAP activation process were studied by isolating and characterizing random multisite mutations. Only a small percentage of mutants have dramatic effects on lac promoter activity. Among the mutations that did affect expression, a 26-fold range in lac promoter activity in vivo was observed in the CAP-independent activity. The highest level of CAP-independent lac expression (13-fold the level of the wild-type lac promoter) correlated with changes in the -40 to -45 sequence and required an intact RNA polymerase alpha subunit for in vitro expression, as expected for an upstream DNA recognition element. Mutant promoters varied in their ability to be stimulated by CAP in vivo, with levels ranging from 2-fold to the wild-type level of 22-fold. Only a change of twofold in responsiveness to CAP could be attributed to direct DNA sequence effects. The -40 to -45 sequence-dependent enhancement of promoter activity and CAP stimulation of promoter activity did not act additively. The mutant promoters also displayed other characteristics, such as the activation of nascent promoter-like activities overlapping lac P1 and, in one case, replicon-dependent changes in promoter activity.

Characterization of dynamics of the psbD light-induced transcription in mature wheat chloroplasts.

Dynamical aspects of three chloroplast promoters responding to change in light condition were examined in mature chloroplasts of wheat (Triticum aestivum) by in vitro transcription. The wheat psbD/C operon has four distinct promoters, two of which named as D/C-3 and D/C-4 promoters dominantly function in mature chloroplasts to produce the mRNAs encoding D2/CP43 and CP43 alone, respectively. Activity of the D/C-3 promoter in mature chloroplasts was reduced to less than 30% by 24 h dark adaptation and recovered by re-illumination to the original level within 30 to 60 min. The activation of the D/C-3 promoter which requires de novo cytoplasmic protein synthesis was induced by low fluence of light (e.g. 16 microE m(-2) s(-1)), but the extent of activation increased with increasing light fluence. The accumulation of mRNAs from the D/C-3 promoter saturated at 2- to 3-fold higher level within 2 h when the dark-adapted seedlings were transferred to the light at 72 microE m(-2) s(-1), concomitant with the increase in rate of D2 synthesis, suggesting that synthesis of D2 in mature chloroplasts is controlled via the D/C-3 promoter activity in a light-dependent way. Activity of the D/C-4 promoter slightly increased in the dark and decreased in the light. Effect of light on the psbA promoter activity was not observed at all in mature chloroplasts. In vitro transcriptional analysis of the D/C-3 promoter with 5' deletion mutations revealed that at least two cis elements which are located within the sequences of -78 to -47 and -46 to -29 of the transcription initiation site, respectively, act as enhancing elements in the D/C-3 promoter. The light-switching element of the transcription, however, was suggested to be located in the core promoter sequence downstream of the -35 element.

HSF recruitment and loss at most Drosophila heat shock loci is coordinated and depends on proximal promoter sequences.

The heat shock response in Drosophila is primarily dependent on the binding of the heat shock transcription factor, HSF, to conserved sequences in heat shock gene promoters, the heat shock elements (HSEs). Here we examine the kinetic relationship of HSF binding to chromosomal loci and heat shock gene transcription in vivo. The features of heat shock promoters that determine the kinetics of HSF binding are also examined. Analyses of HSF association by indirect immunofluorescence with an anti-HSF antibody reveal that fluorescent signals at many loci on polytene chromosomes rapidly increase and then gradually decrease as heat shock time progresses. While overall amounts of fluorescent signal vary from locus to locus, the patterns of acquisition and loss of HSF at most loci are coordinated with only one identified exception. Immunostaining with an anti-RNA polymerase II antibody indicates that the kinetics of RNA polymerase II accumulation on the heat shock loci are similar to those of HSF. Furthermore, nuclear run-on assays confirm that the major heat shock genes are coordinately transcribed during the attenuation period. In contrast, the kinetics of HSF association with HSE "polymers" in a transgenic fly strain are not coordinated with those of endogenous loci. The addition of core promoter sequences to one of the HSEs found in the polymer restores coordinate HSF binding, suggesting that the kinetic patterns of HSF binding depend on a core promoter located near the HSEs. Finally, the distribution of the heat shock protein HSP70 is examined for its role in regulating the attenuated response of HSF to heat shock.

Identification of positive and negative regulatory cis-elements of the rice glutelin Gt3 promoter

A series of 3′ promoter deletions of the rice glutelin Gt3 gene (−181 bp, −278 bp, −300 bp, −320 bp, −482 bp, −580 bp, −775 bp and −835 bp from the transcriptional initiation site) was obtained and linked to the −90 core promoter fragment of the 35S CaMV gene located upstream of the β-glucuronidase protein coding region of pBI 101. After transfer into tobacco via Agrobacterium tumefaciens-mediated transformation, the spatial and temporal of the Gt3 promoter was analyzed in 16- and 24-day-old seeds, as well as leaves and roots of the transgenic tobacco plants. Other than constitutive expression in roots contributed by the CaMV 35S -90 core promoter sequences, significant expression of the intact Gt3-CaMV 35S hybrid promoter was evident only during seed development where maximum expression was evident in 16-day-old seeds. Analysis of hybrid promoters containing various 3′ deletions of the rice promoter revealed two regions, +7 to −181 and −278 to −320, that were essential for maximum Gt3 expression. Removal of the proximal region, +7 to −181, resulted in a 50% decrease of GUS expression with an additional 27% decrease upon removal of the distal region. A negative regulatory element that suppressed expression of the chimeric promoter in leafs is located between −320 bp and −482 bp. These results together with those from an earlier study (Zhao et al., Plant Mol. Biol. 325 (1994) 429–436) from this laboratory demonstrate that the developmental control of the rice Gt3 promoter requires at least three regions governing spatial, temporal and quantitative expression in seeds and at least one region suppressing expression in leaves.

Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B

Background & Aims It was recently reported that two point mutations within the hepatitis B virus (HBV) core promoter region (A to T at position 1762 and G to A at position 1764) are associated with fulminant hepatitis and lead to hepatitis B e antigen (HBeAg)-negative phenotype. The aim of this study was to correlate core promoter sequence variations with HBeAg status and clinical outcome in various forms of HBV infection. Methods Core promoter region of HBV was amplified by polymerase chain reaction and directly sequenced in 94 patients: 37 patients with fulminant hepatitis, 20 with acute self-limited hepatitis, 30 with chronic hepatitis, and 7 patients with end-stage cirrhosis. Results Core promoter region was found to be heterogenous and no specific changes correlated with HBeAg/anti-HBeAg status or survival in patients with fulminant hepatitis. Substitutions at positions 1762 and 1764 were found in HBV strains from 4 patients (10%) with fulminant hepatitis, 2 patients (10%) with self-limited hepatitis, 8 patients (27%) with chronic hepatitis, and in 5 of 7 patients with end-stage cirrhosis. The majority of these patients were HBeAg positive. Conclusions Mutations at positions 1762 and 1764 are rarely observed in HBV strains from patients with fulminant hepatitis B in the United States but are common in patients with chronic hepatitis. Even when present, they seem to be insufficient to lead to the HBeAg-negative phenotype.

Functional promoter and polyadenylation site mapping of the human serotonin (5-HT) transporter gene.

We have isolated and characterized the 5'-flanking region and the proximal polyadenylation site of the human 5-HT transporter gene. The major gene transcript is 2,793 bp in length and it contains 208 bp of 5'-untranslated region (5'-UTR) and 694 bases of 3'-UTR. While only a single mRNA species occurs in rats and mice, the most proximal signal for polyadenylation in the human gene appears to be highly degenerate in comparison to the rat and murine motif. This polyadenylation signal-like motif may lead to alternate usage of additional polyadenylation sites resulting in multiple mRNA species in humans. A TATA-like motif and several potential binding sites for transcription factors including AP1, AP2, SP1, and a cAMP response element (CRE)-like motif are present in the 5'-flanking region. A approximately 1.7 kb fragment beginning 217 bp downstream from the transcription start site, which had been ligated into a luciferase reporter vector and transiently expressed in JAR human placental choriocarcinoma cells, displayed both constitutive and forskolin/cholera toxin-induced promoter activity. Functional promoter mapping revealed that there are negative attenuating elements between bp -1,428 and -1,185 and positive elements between bp -1,184 and -78 from the transcription initiation site. Studies with deletional mutants also indicated that core promoter sequences are contained within 78 bp of the transcription start site and that regulation of cAMP-inducible promoter activity depends on multiple cis-acting elements including two AP1 binding sites and a single CRE-like element located at bp -99. Our findings suggest that (1) the 5-HT transporter gene promoter is active in human JAR cells, but inactive in 5-HT transporter-deficient human SK-N-SH neuroblastoma and HeLa cells, (2) the information contained within 1.4 kb of 5'-flanking sequence is sufficient to confer its cell-specific expression, (3) the promoter responds to cAMP induction, and (4) the expression of the 5-HT transporter gene is regulated by a combination of positive and negative cis-acting elements operating through a basal promoter unit defined by a TATA-like motif.

The RNA Polymerase I Promoter-activating Factor CPBF Is Functionally and Immunologically Related to the Basic Helix-Loop-Helix-Zipper DNA-binding Protein USF

Analysis of the core promoter sequence of the mammalian ribosomal RNA genes revealed an E-box-like sequence (CACGCTG) to which the upstream stimulatory factor (USF) binds. Because the core promoter binding factor (CPBF) binds specifically to the core promoter sequence of the ribosomal RNA gene (Liu, Z., and Jacob, S. T. (1994) J. Biol. Chem. 269, 16618-16626) and resembles USF in some respects, we explored the potential relationship between USF and CPBF. Competition electrophoretic mobility shift assay using labeled core promoter probe and several unlabeled competitor oligonucleotides showed that USF can indeed bind to the core promoter and that only those oligonucleotides with an E-box sequence could compete in the promoter-protein complex formation characteristic of CPBF. This complex formation was thermostable, a unique property of USF. Furthermore, antibodies raised against USF cross-reacted with the 44-kDa component of rat CPBF. To prove further the relationship between CPBF and USF, we examined the effects of the unlabeled USF and ribosomal gene core promoter oligonucleotides on polymerase (pol) I and pol II transcription. Both oligonucleotides inhibited rDNA transcription as well as transcription from the adenovirus major late promoter. Only the oligonucleotides that contain the E-box sequence competed in the transcription. These data indicate that the promoter sequence of mammalian ribosomal RNA gene contains an USF-binding site, that the 44-kDa polypeptide of CPBF is related to the 44/43-kDa polypeptide of human USF, and that USF or USF-related protein can transactivate both pol I and pol II promoters.

A 60-bp core promoter sequence of murine lactate dehydrogenase C is sufficient to direct testis-specific transcription in vitro.

A clone containing the 5' flanking region of the testis-specific murine lactate dehydrogenase C (Ldhc) gene was isolated from a mouse genomic library. Promoter activity was demonstrated within a 720-bp fragment in testis nuclear extract (TN). Interestingly, the addition of liver nuclear extract (LN) significantly repressed Ldhc promoter activity in the transcription assay system. Sequence analysis of this promoter region revealed several ubiquitous cis-regulatory elements, including one TATA box, one GC box, and two putative CCAAT elements. Analysis of a series of deletion mutants revealed that a 60-bp core promoter sequence was sufficient to direct basal, testis-specific transcription in an in vitro transcription system. A 103-kDa protein in TN and a 65-kDa protein in LN bind to the same palindromic sequences within the 60-bp core promoter region.

Context-dependent Effects of Upstream A-Tracts: Stimulation or Inhibition of Escherichia coli Promoter Function

Phased A-tract sequences were inserted in the upstream region of three synthetic promoters known to encompass different rate-limiting steps within the pathway of RNA polymerase-promoter interaction (Ellinger et al., accompanying paper). Promoter PS1, which is rate-limited in complex formation, was stimulated by A-tracts in vivo. Permanganate probing showed that the stimulation is due to an enhanced ability to compete for limiting RNA polymerase in vivo, leading to the increased formation of open complexes. By contrast, promoters PS2 and PS3, which are rate-limited in steps following open complex formation, were inhibited in vivo by A-tracts. Permanganate probing showed that the inhibition was accompanied by an A-tract-dependent accumulation of stalled initial transcribing complexes. A single A-tract was as effective as three.The phasing of the A-tracts with respect to the core promoter sequence was found to be important for promoter function. The position that caused maximal activation at one promoter caused maximal inhibition at another. These results suggest that the same molecular interaction gives rise to both inhibition and activation. This is likely to be due to facilitated RNA polymerase binding in the presence of A-tracts, which stimulates binding-limited promoters but inhibits promoter function in which polymerase escape and promoter clearance is rate limiting.

Partitioning of plasmid R1 Ten direct repeats flanking the parA promoter constitute a centromere-like partition site parC, that expresses incompatibility

The parA partitioning system of plasmid R1 consists of three different components: the cis-acting Centromere-like parC site, and the two trans-acting proteins Parts and ParR. These three components are contained within a region of 1·6 kb. The parC site is located upstream of the two genes, parts and parR, which are expressed as an operon from the parA promoter. The parC site contains an array of ten 11 base-pair direct repeats, organized in two sets of five repeats flanking the parA core promoter sequences. Deletions and point mutations were introduced in the parA locus, resulting in partially stable and unstable plasmids. An analysis of these parA − plasmids showed that Parts and ParR are transacting. The 160 by minimal parC region contained sufficient in cis information for efficient trans-complementation. Both proteins were required for maximal stabilization of a parC + mini-R1 plasmid, although ParR alone, donated either in cis or in trans, yielded partial stabilization. Plasmids that overexpressed ParR caused destabilization of a co-resident parA + plasmid, whereas overexpression of Parts had no such effect. The parC site exerted incompatibility ( incA) at high but not at low copy number. Likewise, the entire parA system exerted incompatibility in a copy number-dependent fashion, and stronger than the incompatibility expressed by parC alone.

Partitioning of plasmid R1 The parA operon is autoregulated by parR and its transcription is highly stimulated by a downstream activating element

The parA partitioning system of plasmid RI mediates efficient stabilization of R1 and F-derived replicons. The parA system is encoded by a continuous DNA segment of approximately 1600 base-pairs and consists of three components. Two adjacent genes, parM and parR, coding for the trans-acting proteins ParM and ParR, and the cis-acting parC site. The centromere-like parC site is located upstream of parM and parR and contains the parA promoter. The parM and parR genes are co-transcribed as an operon from the parA promoter. The 5′ end of the parA encoded transcript was mapped to the center of the parC region at +115. The −10 and −35 core promoter sequences are flanked by the two sets of five direct repeats in parC (the ParR boxes). The parA promoter was found to be negatively regulated by the parR gene product, whereas the parM gene product seemingly was not involved in the regulation. Surprisingly, a region downstream of the parA promoter enhanced transcription from the promoter many-fold (30 to 50-fold). The parC site titrated the ParR protein, suggesting that the ParR protein interacts directly with the parC site. Using an engineered parA system we found that the parC site could be complemented in cis by the parM and parR genes. Furthermore, the proper function of the parC site was highly dependent on the expression level of ParM and ParR. The incompatibility associated with the parC site could not be suppressed by overexpression of the ParM and ParR proteins. Based on these results we suggest a novel partition model involving pairing of newly replicated plasmid molecules.

Recognition of the Xenopus ribosomal core promoter by the transcription factor xUBF involves multiple HMG box domains and leads to an xUBF interdomain interaction.

The interaction of the ribosomal transcription factor xUBF with the RNA polymerase I core promoter of Xenopus laevis has been studied both at the DNA and protein levels. It is shown that a single xUBF-DNA complex forms over the 40S initiation site (+1) and involves at least the DNA sequences between -20 and +60 bp. DNA sequences upstream of +10 and downstream of +18 are each sufficient to direct complex formation independently. HMG box 1 of xUBF independently recognizes the sequences -20 to -1 and +1 to +22 and the addition of the N-terminal dimerization domain to HMG box 1 stabilizes its interaction with these sequences approximately 10-fold. HMG boxes 2/3 interact with the DNA downstream of +22 and can independently position xUBF across the initiation site. The C-terminal segment of xUBF, HMG boxes 4, 5 or the acidic domain, directly or indirectly interact with HMG box 1, making the core promoter sequences between -11 and -15 hypersensitive to DNase. This interaction also requires the DNA sequences between +17 and +32, i.e. the HMG box 2/3 binding site. The data suggest extensive folding of the core promoter within the xUBF complex.

The function of conserved elements in the promoter of the mouse angiotensinogen gene.

The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a mini-gene containing 750 bp of 5'-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into nonhepatic cell lines. We found that 5'-deletion mutant constructs, containing sequences from +25 to -90 bp and -321 to -750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Sp1-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5' to -244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.

A 3'-coterminal nested set of independently transcribed mRNAs is generated during Berne virus replication

By using poly(A)-selected RNA from Berne virus (BEV)-infected embryonic mule skin cells as a template, cDNA was prepared and cloned in plasmid pUC9. Recombinants covering a contiguous sequence of about 10 kilobases were identified. Northern (RNA) blot hybridizations with various restriction fragments from these clones showed that the five BEV mRNAs formed a 3'-coterminal nested set. Sequence analysis revealed the presence of four complete open reading frames of 4743, 699, 426, and 480 nucleotides, with initiation codons coinciding with the 5' ends of BEV RNAs 2 through 5, respectively. By using primer extension analysis and oligonucleotide hybridizations, RNA 5 was found to be contiguous on the consensus sequence. The transcription of BEV mRNAs was studied by means of UV mapping. BEV RNAs 1, 2, and 3 were shown to be transcribed independently, which is also likely--although not rigorously proven--for RNAs 4 and 5. Upstream of the AUG codon of each open reading frame a conserved sequence pattern was observed which is postulated to function as a core promoter sequence in subgenomic RNA transcription. In the area surrounding the core promoter region of the two most abundant subgenomic BEV RNAs, a number of homologous sequence motifs were identified.