Transcriptional Regulatory Sequences Research Articles

Microarray Transfection Analysis of cAMP and cGMP Regulation of Gene Expression

Transcription factor activation by phosphorylation is a common mechanism for both developmental and hormonal control of gene expression. Over 2000 transcription factors and 600 protein kinases are predicted from the human genome sequence but only a small fraction of the potential regulatory interactions have been experimentally tested. We have defined transfection conditions to functionally assess these interactions in a microarray transfection format. The transfection method employed is termed Surface Transfection and Expression Protocol (STEP) and relies on the production of recombinant polyfunctional proteins to mediate the high efficiency in situ transfection of different cell types. Cis-acting transcriptional regulatory sequences driving the expression of GFP and other reporters were used to probe the activity of specific transcription factor families in the presence or absence of transfected protein kinases. We find that regulation of native promoters such as the c-fos promoter by cAMP- and cGMP-dependent protein kinases is controlled by conserved non-genic (CNG) sequences surrounding the c-fos gene. In particular, an evolutionarily conserved sequence within the first intron of the c-fos gene plays a key role in transcriptional regulation by these two protein kinases. The regulation by the CNG sequences was promoter specific in most cases. This microarray transfection approach should be useful in defining novel pathways by which protein phosphorylation regulates transcription factor activity as well as other functional interactions in vivo. This work was supported by NIH grants HG002367 and DK063340.

An improved mouse line for Cre-induced cell ablation due to diphtheria toxin A, expressed from the Rosa26 locus

The means to specifically ablate cells inside of a living organism have recently been improved and facilitated by stable mouse lines, carrying conditional expression constructs for diphtheria toxin (DT) or diphtheria toxin receptor, that could be activated upon Cre-mediated recombination or the application of diphtheria toxin, respectively. We have lately described the R26:lacZ/DT-A line (Brockschnieder et al., 2004, Mol Cell Biol 24:7636-7642) in which a loxP-conditional DTA allele was introduced into the ubiquitously expressed Rosa26 locus. This strain allowed the ablation of a wide spectrum of cell types by crossing it to tissue specific Cre lines. Unexpectedly, homozygous (but not heterozygous) animals of the R26:lacZ/DT-A line developed some degenerative abnormalities in a variety of tissues. The defects were most probably caused by leaky expression of small amounts of toxin from the unrecombined lacZ(flox)DT-A cassette. Here we show that insertion of an additional transcriptional regulatory sequence (bovine growth hormone polyadenylation signal, bpA) following the lacZ open reading frame prevented the formation of any defects in homozygous mice. The modification did not affect the functionality of the lacZ(flox)DTA allele, as exemplified by the complete ablation of oligodendrocytes upon Cre-mediated recombination. The novel R26:lacZbpA(flox)DTA line is expected to greatly facilitate the reliable generation of cell type ablated mice.

Complex formations involving both SP-1 and SP-3 at the transcriptional regulatory sequence correlate with the activation of the Keratin 14 gene in human oral squamous cell carcinoma cells

We have previously reported that significantly higher levels of Keratin 14 (Ker-14) was observed in oral squamous cell carcinoma (OSCC) and severely dysplastic tissues, whereas this expression was reversed in hyperplasia and in mild to moderate dysplasia. In this study, the mechanism of Keratin 14 activation in oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3 and Ca9-22) was investigated. Reporter analysis demonstrated that an upstream region (-1759/-1629) accounted for efficient promoter activity. Furthermore, electromobility sift and supershift assay demonstrated that interactions of the SP-1/SP-3 complex at the elements resided in -1737/-1702 and -1680/-1652 and may be essential for this activation in OSCC cells.

Effects of the DNA Methylase Inhibitor Zebularine on Global Gene Expression in AML1-ETO-Positive Acute Myeloid Leukemia.

In acute myeloid leukemias (AML) containing the t(8;21)(q22;q22) translocation, the RUNX1 gene, encoding the transcription factor AML1, is fused to RUNX1T1 (ETO), resulting in a protein that retains the DNA binding domain of AML1 and, via ETO-mediated recruitment of corepressor complexes, represses AML1-dependent gene transcription. Because the recruited corepressor complex contains an active histone deacetylase (HDAC), and HDAC-mediated chromatin modifications are dynamically linked to DNA methylation, we were interested in defining the scope of epigenetic gene silencing in AML1-ETO-positive leukemias. To assess this, we examined the effects of zebularine, a newer DNA methylase inhibitor, on global gene expression in the AML1-ETO-positive AML cell line, SKNO-1. In a first step, we demonstrated the efficacy of zebularine treatment by confirming upregulation of CDKN2B, a gene known to be repressed by promoter methylation in AML1-ETO-positive AML. Likewise, the degree of methylation of the AKAP12 promoter, as measured by bisulfite sequencing, was reduced following treatment. We also examined the toxicity of zebularine in SKNO-1 and observed dose-dependent growth inhibition of cultures, owing to an increase in apoptosis (annexin V positivity) and decrease in viability (propidium iodide staining). Zebularine did not induce differentiation (CD11b expression) or alter the cell cycle (DNA analysis by flow cytometry). Using Affymetrix U133A arrays, we next generated triplicate genome-wide expression profiles of untreated or zebularine-treated SKNO-1 blasts. In parallel, we also obtained profiles of SKNO-1 cells treated with 5-aza-deoxycytidine (5-aza-dC), as well as expression profiles of a second AML1-ETO-positive leukemic cell line, Kasumi-1SJ, treated with zebularine. Surprisingly, zebularine-induced changes in gene expression were distinct from the expression changes induced by 5-aza-dC, indicating significant differences in the effects of these putative demethylating agents on leukemia cells.To identify a signature of genes that might be epigenetically repressed in AML1-ETO-positive leukemias, we compared the untreated to treated replicate cultures within each cell line/drug combination using statistical approaches. Again, the lists of selected transcripts showed little overlap between zebularine and 5-aza-dC. The genes induced following drug treatment included a cell cycle regulator (CCND3), developmental transcription regulators (ID2, HOXA5), a membrane protein possibly involved in myeloma progression (LAPTM5), and genes functionally related to apoptosis (LGALS1, TNFSF10, SIPA1), protein /nucleic acid catabolism (PSMC3, RNASE2) or cellular defense mechanisms (EPHX1, TYROBP, TRGV9). We assessed the methylation status of the identified 5′ CpG islands of CCND3, ID2, HOXA5 and an intronic regulatory region of LAPTM5 in SKNO-1 cells, primary blasts from pediatric AML patients, or normal peripheral blood leukocytes. Remarkably, no evidence of aberrant methylation was found. From these data we conclude:The cellular effects of zebularine and 5-aza-dC are more divergent than previously appreciated.The observed changes in expression of CCND3, ID2, HOXA5 and LAPTM5 are likely due to methylation processes in yet undefined regulatory regions, or are the result of other transcriptional regulatory processes unrelated to changes in the methylation status of the transcriptional regulatory sequences of these genes.In the two cell lines examined, a common AML1-ETO-dependent methylation “signature” does not exist.

Antiviral activity of morpholino oligomers designed to block various aspects of Equine arteritis virus amplification in cell culture

The antiviral efficacy of ten antisense phosphorodiamidate morpholino oligomers (PMOs) directed against Equine arteritis virus (EAV), a nidovirus belonging to the family Arteriviridae, was evaluated in mammalian (Vero-E6) cells. Peptide-conjugated PMOs (P-PMOs) supplied in cell culture medium at micromolar concentrations were efficiently taken up by Vero-E6 cells and were minimally cytotoxic. The P-PMOs were designed to base pair to RNA sequences involved in different aspects of EAV amplification: genome replication, subgenomic mRNA synthesis, and translation of genome and subgenomic mRNAs. A novel recombinant EAV, expressing green fluorescent protein as part of its replicase polyproteins, was used to facilitate drug screening. A moderate reduction of EAV amplification was observed with relatively high concentrations of P-PMOs designed to anneal to the 3'-terminal regions of the viral genome or antigenome. To determine if the synthesis of subgenomic mRNAs could be specifically reduced, transcription-regulating sequences essential for their production, but not for the production of genomic RNA, were targeted, but these P-PMOs were found to be ineffective at transcription interference. In contrast, all four P-PMOs designed to base pair with targets in the genomic 5' untranslated region markedly reduced virus amplification in a sequence-specific and dose-responsive manner. At concentrations in the low micromolar range, some of the P-PMOs tested completely inhibited virus amplification. In vitro translation assays showed that these P-PMOs were potent inhibitors of translation. The data suggest that these compounds could be useful as reagents for exploring the molecular mechanics of nidovirus translation and have anti-EAV potential at relatively low concentrations.

Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers

The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5' untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection.

Torovirus non-discontinuous transcription: mutational analysis of a subgenomic mRNA promoter.

Toroviruses (order Nidovirales) are enveloped positive-strand RNA viruses of mammals. The prototype torovirus, equine torovirus strain Berne (Berne virus [BEV]), uses two different transcription strategies to produce a 3'-coterminal nested set of subgenomic (sg) mRNAs. Its mRNA 2 carries a leader sequence derived from the 5' end of the genome and is produced via discontinuous transcription. The remaining three sg mRNAs, 3 to 5, are colinear with the 3' end of the genome and are made via non-discontinuous RNA synthesis. Their synthesis is supposedly regulated by short conserved sequence motifs, 5'-ACN3-4CUUUAGA-3', within the noncoding intergenic regions that precede the M, HE, and N genes (A. L. van Vliet, S. L. Smits, P. J. Rottier, and R. J. de Groot, EMBO J. 21:6571-6580, 2002). We have now studied the--for nidoviruses unusual--non-discontinuous transcription mechanism in further detail by probing the role of the postulated transcription-regulating sequences (TRSs). To this end, we constructed a synthetic defective interfering (DI) RNA, carrying a 24-nucleotide segment of the intergenic region between the HE and N genes. We demonstrate that this DI RNA, when introduced into BEV-infected cells, directs the synthesis of a sg DI RNA species; in fact, a 16-nucleotide cassette containing the TRS already proved sufficient. Synthesis of this sg DI RNA, like that of mRNAs 3 to 5 of the standard virus, initiated at the 5'-most adenylate of the TRS. An extensive mutational analysis of the TRS is presented. Our results provide first and formal experimental evidence that the conserved motifs within the BEV intergenic sequences indeed drive sg RNA synthesis.

Evolutionary analysis of the well characterized endo16 promoter reveals substantial variation within functional sites

The evolutionary mechanisms that operate on genetic variation within transcriptional regulatory sequences are not well understood. We present here an evolutionary analysis of an exceptionally well characterized cis-regulatory region, the endo16 promoter of the purple sea urchin. Segregating variation reveals striking differences in the intensity of negative selection among regulatory modules, reflecting their distinct functional roles. Surprisingly, transcription-factor-binding sites are as polymorphic and as likely to contain fixed differences as flanking nucleotides. Whereas nucleotides in protein-binding sites in the most proximal regulatory module exhibit reduced variation, those in other modules tend to be more polymorphic than putatively nonfunctional nucleotides. Two unrelated large insertions at the same position within the promoter are segregating at low frequencies; one is a strong ectodermal repressor that contains 16 verified transcription-factor-binding sites. These results demonstrate that a simple relationship between conservation and function does not exist within this cis-regulatory region and highlight significant population heterogeneity in the fine structure of a well understood promoter.

Cis-Regulatory Logic of Short-Range Transcriptional Repression in Drosophila melanogaster

Bioinformatics analysis of transcriptional control is guided by knowledge of the characteristics of cis-regulatory regions or enhancers. Features such as clustering of binding sites and co-occurrence of binding sites have aided enhancer identification, but quantitative predictions of enhancer function are not yet generally feasible. To facilitate the analysis of regulatory sequences in Drosophila melanogaster, we identified quantitative parameters that affect the activity of short-range transcriptional repressors, proteins that play key roles in development. In addition to the previously noted distance dependence, repression is strongly influenced by the stoichiometry, affinity, spacing, and arrangement of activator binding sites. Repression is insensitive to the type of activation domain, suggesting that short-range repression may primarily affect activators at the level of DNA binding. The activity of several short-range, but not long-range, repressors is circumscribed by the same quantitative parameters. This cis-regulatory "grammar" may aid the identification of enhancers regulated by short-range repressors and facilitate bioinformatic prediction of the functional output of transcriptional regulatory sequences.

Regulation of Expression of the Chorionic Gonadotropin/Luteinizing Hormone Receptor Gene in the Human Myometrium: Involvement of Specificity Protein-1 (Sp1), Sp3, Sp4, Sp-Like Proteins, and Histone Deacetylases

At present there is little information on the regulatory processes by which the chorionic gonadotropin (CG)/LH receptor gene is regulated in the human myometrium during pregnancy and labor. Employing human primary myometrial cell cultures in conjunction with DNA affinity purification assays/Western analysis, DNA binding studies, CG/LH promoter luciferase reporter gene deletion constructs in transfection assays, and measurement of endogenous mRNA levels in vivo by duplex RT-PCR, we have determined the role that the major transcriptional regulatory sequences from the +1 ATG codon to -2678 bp play in modulating expression of the CG/LH receptor gene in the myometrium. We report that the distal -180 to -2678 bp region of the promoter, although capable of binding members of the Jun family via the multiple activator protein-1 sites within this region, has no significant role in regulating the expression of the CG/LH receptor gene in myometrial cells. In contrast, the two specificity protein-1 to -4 (Sp1-4) GC boxes within the +1 to -180 bp proximal promoter are central to expression of the gene in the myometrium. However, not only are Sp1/Sp3 proteins involved in this process, but Sp4 and a novel Sp-like factor(s) also have an intimate part in transcriptional regulation of the gene. It would appear that Sp1/Sp3/Sp4 and Sp-like proteins are involved in recruiting histone deacetylase complexes to the proximal promoter, preventing chromatin remodeling resulting in transcriptional repression of the gene. Our data suggest that administration of the histone deacetylase inhibitor trichostatin A to human myometrial cells in vitro and in vivo substantially removes this silencing effect on expression of the gene and may implicate the use of this and similar agents in increasing myometrial CG/LH receptor levels and subsequent maintenance of uterine relaxation during fetal maturation.

Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis.

The generation of subgenomic mRNAs in coronavirus involves a discontinuous mechanism of transcription by which the common leader sequence, derived from the genome 5' terminus, is fused to the 5' end of the mRNA coding sequence (body). Transcription-regulating sequences (TRSs) precede each gene and include a conserved core sequence (CS) surrounded by relatively variable sequences (5' TRS and 3' TRS). Regulation of transcription in coronaviruses has been studied by reverse-genetics analysis of the sequences immediately flanking a unique CS in the Transmissible gastroenteritis virus genome (CS-S2), located inside the S gene, that does not lead to detectable amounts of the corresponding mRNA, in spite of its canonical sequence. The transcriptional inactivity of CS-S2 was genome position independent. The presence of a canonical CS was not sufficient to drive transcription, but subgenomic synthesis requires a minimum base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) provided by the CS and its adjacent nucleotides. A good correlation was observed between the free energy of TRS-L and cTRS-B duplex formation and the levels of subgenomic mRNA S2, demonstrating that base pairing between the leader and body beyond the CS is a determinant regulation factor in coronavirus transcription. In TRS mutants with increasing complementarity between TRS-L and cTRS-B, a tendency to reach a plateau in DeltaG values was observed, suggesting that a more precise definition of the TRS limits might be proposed, specifically that it consists of the central CS and around 4 nucleotides flanking 5' and 3' the CS. Sequences downstream of the CS exert a stronger influence on the template-switching decision according to a model of polymerase strand transfer and template switching during minus-strand synthesis.

Fluorescent Proteins Expressed in Mouse Transgenic Lines Mark Subsets of Glia, Neurons, Macrophages, and Dendritic Cells for Vital Examination

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.

Herpes Simplex Virus Type 1 Amplicons and their Hybrid Virus Partners, EBV, AAV, and Retrovirus

HSV-1 amplicons can accommodate foreign DNA of any size up to 150 kbp. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, or multiple transgenes can be inserted in a modular fashion. HSV-1 amplicon vectors deliver DNA efficiently into the cell nucleus as an extrachromosomal, non-replicating circular concatenate, which is rapidly diluted, at least in dividing cells. Consequently, transgene expression is lost within days to weeks in dividing cells, but may be retained for months in non-dividing cells. In contrast, vectors based on Epstein-Barr virus, adeno-associated virus, or retroviruses can mediate long-term transgene expression, as vector DNA is retained by episomal replication or chromosomal integration. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, thereby conserving the large transgene capacity of HSV-1, and genetic elements from other viruses that confer genetic stability to the vector DNA within transduced cells. Additional strategies to sustain genetic material in infected cells include the incorporation of recombinases from different bacteriophages or transposable elements of the Tc1/mariner family in the amplicon vector. Moreover, modification of the HSV-1 virion itself offers a myriad of possibilities to improve gene delivery by targeting specific cell populations or transporting foreign proteins, such as Cre recombinase or the adeno-associated virus Rep protein, which can control the fate and expression of the therapeutic transgene.

Regulatory sequences of H19 and IGF2 genes in DNA‐based therapy of colorectal rat liver metastases

Malignant tumors of the liver are among the most common causes of cancer-related death throughout the world. Current therapeutic approaches fail to control the disease in most cases. This study seeks to explore the potential utility of transcriptional regulatory sequences of the H19 and insulin growth factor 2 (IGF2) genes for directing tumor-selective expression of a toxin gene (A fragment of diphtheria toxin), delivered by non-viral vectors. The therapeutic potential of the toxin vectors driven by the H19 and the IGF2-P3 regulatory sequences was tested in a metastatic model of rat CC531 colon carcinoma in liver. Intratumoral injection of these vectors into colon tumors implanted in the liver of rats induced an 88% and a 50% decrease respectively in the median tumor volume as compared with the control groups. This therapeutic action was accompanied by increased necrosis of the tumor. Importantly, no signs of toxicity were detected in healthy animals after their treatment by the toxin expression vectors. DT-A was preferentially expressed in liver metastases after being transfected with H19 or IGF2-P3 promoter-driven DT-A expression plasmids, causing a very significant inhibition of tumor growth as a result of its cytotoxic effect. Our findings strongly support the feasibility of our proposed therapeutic strategy, which may contribute to open new gene therapeutic options for human liver metastases.

Selecting transpositions using phage P1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and P1-derived artificial chromosomes

New Tn10 minitransposons were constructed to functionally map long-range transcription regulatory sequences in bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs). Each contained a wild-type loxP site but, significantly, contained no mammalian or bacterial genes and/or promoter elements within the transposed portion of DNA. In contrast to loxP transposons described previously, the new ones do not introduce transcription regulatory elements capable of interfering with those endogenous to the BAC clone in functional mapping studies. Progressive deletions from the loxP end of genomic DNA were efficiently generated using these transposons, and a series of truncations generated in a green fluorescence protein (GFP)–BAC fusion clone unambiguously identified three new long-range enhancer sequences functionally in the Nkx2-5 gene in transgenic mice. Insertions of these new transposons lacking antibiotic resistance genes into a BAC or PAC were indirectly selected by their ability to delete enough DNA from the clone so as to enable its packaging within a P1 phage head with both loxP sites intact for subsequent recovery of the large plasmid. The outcome of such an indirect mode of selection is both desirable and undesirable. First, because the screen is not antibiotic resistance marker dependent, the same transposon can be used to generate nested deletions efficiently in both BACs and PACs. Second, deletions through intrainsert recombinations unrelated to loxP/Cre also get packaged and recovered, and size analyses of the BAC/PAC vector band after NotI digestion is indispensable to identify authentic loxP/Cre deletions. The procedure nevertheless offers a potential approach to map recombinogenic sequences in BACs and PACs.

Non-classical regulation of estrogen receptor-α by ICI182,780

Estrogen receptor-α (ERα) regulates transcription through a number of molecular mechanisms. Two mechanisms by which ERα acts directly in the nucleus have emerged: (1) in classical ERα action, estrogen-bound receptor binds estrogen response elements (ERE) and regulates promoters by recruiting coactivators or corepressors to DNA; (2) non-classical action is not dependent on ERα binding to EREs; its mechanism is not as clearly defined as classical action. In many instances, non-classical action is mediated by tethering of ERα to other DNA-binding proteins, facilitating recruitment of coregulators to transcription regulatory sequences. In some cell types, non-classical stimulation can be enhanced by antagonists and repressed by agonists of ERα. Here, we show that non-classical action of ERα in 293 cells occurs in a wide range of enhancers and enhancer binding proteins. ERα stimulates AP-1 elements, cyclic AMP response elements (CRE), and serum response elements (SRE) in the presence of the antiestrogen ICI182,780. Further, in the presence of ICI182,780, ERα stimulates activation domains of Jun, ATF-2, Elk, and CRE-binding protein (CREB). Non-classical ERα regulation described here does not appear to be sensitive to point mutations which affect classical and tethered ERα action; moreover, in our experiments, non-classical action is uniquely sensitive to nuclear transport inhibition by leptomycin B. Because ICI182,780 appears to affect multiple and diverse transcriptional systems, our results are likely explained by ERα-dependent modulation of common components of the transcriptional machinery and may not be completely explained by tethering of ERα to specific transcription factors.

Complete sequences of 3′ end coding region for structural protein genes of turkey coronavirus

Overlapping fragments of genomic RNA spanning 6963 nucleotides from 5′ end of spike (S) protein gene to 3′ end of nucleocapsid (N) protein gene of turkey coronavirus (TCoV) were amplified by reverse-transcription-polymerase chain reaction (RT-PCR). The primers were derived from the corresponding sequences of infectious bronchitis virus (IBV). The PCR products were cloned and sequenced and their nucleic acid structure and similarity to published sequences of other coronaviruses were analyzed. Sequencing and subsequent analysis revealed 9 open reading frames (ORFs) representing the entire S protein gene, tricistronic gene 3, membrane (M) protein gene, bicistronic gene 5, and N protein gene in the order of 5′–3′. The overall nucleic acid structures of these encoding regions of TCoV were very similar to the homologous regions of IBV. The consensus transcription-regulating sequence (TRS) of IBV, CT(T/G)AACAA, was highly conserved in TCoV genome at the levels of nucleotide sequence and location in regarding to the initiation codon of individual genes. Pair-wise comparison of gene 3, M gene, gene 5, or N gene sequences with their counterparts of IBV revealed high levels (82.1–92.0%) of similarity. Phylogenetic analysis based on the deduced amino acid sequences of S, M, or N protein demonstrated that TCoV was clustered within the same genomic lineage as the IBV strains while all the other mammalian coronaviruses were grouped into separate clusters corresponding to antigenic groups I or II. There were substantial differences of S protein sequence between TCoV and IBV with only 33.8–33.9% of similarity.

Regulation of relative abundance of arterivirus subgenomic mRNAs.

The subgenomic (sg) mRNAs of arteriviruses (order Nidovirales) form a 5'- and 3'-coterminal nested set with the viral genome. Their 5' common leader sequence is derived from the genomic 5'-proximal region. Fusion of sg RNA leader and "body" segments involves a discontinuous transcription step. Presumably during minus-strand synthesis, the nascent RNA strand is transferred from one site in the genomic template to another, a process guided by conserved transcription-regulating sequences (TRSs) at these template sites. Subgenomic RNA species are produced in different but constant molar ratios, with the smallest RNAs usually being most abundant. Factors thought to influence sg RNA synthesis are size differences between sg RNA species, differences in sequence context between body TRSs, and the mutual influence (or competition) between strand transfer reactions occurring at different body TRSs. Using an Equine arteritis virus infectious cDNA clone, we investigated how body TRS activity affected sg RNA synthesis from neighboring body TRSs. Flanking sequences were standardized by head-to-tail insertion of several copies of an RNA7 body TRS cassette. A perfect gradient of sg RNA abundance, progressively favoring smaller RNA species, was observed. Disruption of body TRS function by mutagenesis did not have a significant effect on the activity of other TRSs. However, deletion of body TRS-containing regions enhanced synthesis of sg RNAs from upstream TRSs but not of those produced from downstream TRSs. The results of this study provide considerable support for the proposed discontinuous extension of minus-strand RNA synthesis as a crucial step in sg RNA synthesis.

Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires both G-box and GCC-motif elements

Putrescine N-methyltransferase (PMT) catalyzes the first committed step of nicotine biosynthesis, converting putrescine into N-methylputrescine. A variety of chemical, environmental, and developmental cues have been implicated in its regulation. Here we have examined the differential expression of beta-glucuronidase (GUS) transgenes under the control of the transcriptional regulatory sequences of four distinct members of the NtPMT gene family from tobacco (Nicotiana tabacum L.). BY-2 cell cultures expressing various NtPMT promoter-GUS constructs were examined for their response to treatment with various combinations of methyl jasmonate (MeJA), auxin (AUX), and ethylene (ETH). All four NtPMT gene promoters examined were inducible by MeJA, although the extent of the induction varied dramatically, with the NtPMT1a promoter being the most responsive. High AUX levels in the cell growth media repressed NtPMT::GUS transgene expression and inhibited their MeJA-induced transcription. Treatment of BY-2 cells with ETH alone did not result in a significant alteration in NtPMT::GUS expression. However, similar to AUX, ETH treatment led to the suppression of MeJA-induced transcription. Detailed deletion analysis of the NtPMT1a gene promoter showed that as little as 111 bp upstream of the transcriptional start site were sufficient to confer MeJA-responsiveness. Deletion of a conserved G-box element (GCACGTTG) at -103 to -96 bp completely abolished MeJA-responsiveness. Further mutagenesis studies revealed that in addition to a functional G-box, MeJA-responsiveness of the NtPMT1a promoter also required a TA-rich region and a GCC-motif (TGCGCCC) located at -80 to -69 bp and -62 to -56 bp relative to the start site, respectively. A synthetic G-box tetramer (4 X syn G-box) fused to a -83 bp fragment from the NtPMT1a promoter (containing the TA-rich region, GCC-box, and TATA-box) displayed a 30-fold induction by MeJA treatment, whereas when the 4 X syn G-box was fused to a minimal (-46 bp) promoter fragment derived from the CaMV 35S gene, no induction by MeJA treatment was detected. Our results indicate that multiple intersecting signal transduction pathways and different transcriptional regulatory factors are involved in mediating JA-responsiveness of NtPMT expression in tobacco.

626. A Novel Prostate-Specific Adenovirus for Gene Expression in Hormone-Sensitive and Hormone-Refractory Prostate Cancer Cells

To develop a transcriptional regulatory sequence for prostate-specific transgene expression that is more potent then the ones currently used in experimental and clinical prostate cancer gene therapy, we constructed various recombinant expression control sequences comprising the TARP promoter (TARPp) and the PSA enhancer (PSAe) and/or the PSMA enhancer (PSMAe). Transcriptional activities of the expression control sequences were examined by luciferase reporter gene assays on transiently transfected cells. We found that gene expression controlled by a chimeric regulatory sequences comprising PSAe linked to PSMAe, in turn linked to TARPp (PPT) is highly prostate-specific and active in prostate cancer cells both in the presence and absence of testosterone. Since prostate cancer patients are often treated by androgen withdrawal, it may be beneficial to the patient to use a regulatory sequence that results in high transgene expression also in the absence of testosterone. Recombinant adenoviruses with the PPT sequence in the E1 position of an E1/E3-deleted adenovirus serotype 5 vectors were constructed. A DNA insulator from the mouse H19 imprinting control region was inserted directly upstream of the expression cassette to protect the transcriptional integrity of PPT from interfering adenoviral sequences. The produced viruses confer prostate-specific transgene expression when screened on cell lines of various origins. We believe that an adenovirus with E1A and/or other therapeutic gene expression, controlled by the H19 insulator-shielded PPT regulatory sequence, is a promising candidate for prostate cancer gene therapy.