Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine Decarboxylase Promoters from Opium Poppy Are Conserved in Transgenic Tobacco1

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

Transcription from Heterologous rRNA Operon Promoters in Chloroplasts Reveals Requirement for Specific Activating Factors1

The plastid rRNA (rrn) operon in chloroplasts of tobacco (Nicotiana tabacum), maize, and pea is transcribed by the plastid-encoded plastid RNA polymerase from a ς70-type promoter (P1). In contrast, the rrn operon in spinach (Spinacia oleracea) and mustard chloroplasts is transcribed from the distinct Pc promoter, probably also by the plastid-encoded plastid RNA polymerase. Primer-extension analysis reported here indicates that in Arabidopsis both promoters may be active. To understand promoter selection in the plastid rrn operon in the different species, we have tested transcription from the spinach rrn promoter in transplastomic tobacco and from the tobacco rrn promoter in transplastomic Arabidopsis. Our data suggest that transcription of the rrn operon depends on species-specific factors that facilitate transcription initiation by the general transcription machinery.

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.

Deregulation of PAX-5 by translocation of the Emu enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma.

Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene revealed that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A previous molecular analysis of a t(9;14) breakpoint from a diffuse large-cell lymphoma (KIS-1) demonstrated that the immunoglobulin heavy-chain (IgH) locus on 14q32 was juxtaposed to chromosome 9p13 sequences of unknown function [Ohno, H., Furukawa, T., Fukuhara, S., Zong, S. Q., Kamesaki, H., Shows, T. B., Le Beau, M. M., McKeithan, T. W., Kawakami, T. & Honjo, T. (1990) Proc. Natl. Acad. Sci. USA 87,628-632]. Here we show that the KIS-1 translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity of the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation.

Two Arabidopsis cyclin promoters mediate distinctive transcriptional oscillation in synchronized tobacco BY-2 cells.

Cyclins are cell cycle regulators whose proteins oscillate dramatically during the cell cycle. Cyclin steady-state mRNA levels also fluctuate, and there are indications that both their rate of transcription and mRNA stability are under cell cycle control. Here, we demonstrate the transcriptional regulation of higher eukaryote cyclins throughout the whole cell cycle with a high temporal resolution. The promoters of two Arabidopsis cyclins, cyc3aAt and cyc1At, mediated transcriptional oscillation of the beta-glucuronidase (gus) reporter gene in stably transformed tobacco BY-2 cell lines. The rate of transcription driven by the cyc3aAt promoter was very low during G1, slowly increased during the S phase, peaked at the G2 phase and G2-to-M transition, and was down-regulated before early metaphase. In contrast, the rate of the cyc1At-related transcription increased upon exit of the S phase, peaked at the G2-to-M transition and during mitosis, and decreased upon exit from the M phase. This study indicates that transcription mechanisms that seem to be conserved among species play a significant role in regulating the mRNA abundance of the plant cyclins. Furthermore, the transcription patterns of cyc3aAt and cyc1At were coherent with their slightly higher sequence similarity to the A and B groups of animal cyclins, respectively, suggesting that they may fulfill comparable roles during the cell cycle.

Krüppel-associated box-mediated repression of RNA polymerase II promoters is influenced by the arrangement of basal promoter elements.

The evolutionarily conserved Krüppel-associated box (KRAB) is present in the N-terminal regions of more than one-third of all Krüppel-class zinc finger proteins. Recent experiments have demonstrated that the KRAB-A domain tethered to a promoter DNA by connecting to heterologous DNA-binding protein domain or targeted to a promoter-proximal RNA sequence acts as a transcriptional silencing of RNA polymerase II promoters. Here we show that expression of KRAB domain suppresses in vivo the activating function of various defined activating transcription factors, and we demonstrate that the KRAB domain specifically silences the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription initiation is directed by a pyrimidine-rich initiator element, however, are relatively unaffected. We also report in vitro transcription experiments indicating that the KRAB domain is able to repress both activated and basal promoter activity. Thus, the KRAB domain appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.

A mammalian arginine/lysine transporter uses multiple promoters.

The mCAT-2 gene encodes a Na(+)-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system y+. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA transport in Xenopus oocytes. The two genes differ in their tissue expression and induction patterns. Here we report the presence of diverse 5' untranslated region (UTR) sequences in mCAT-2 transcripts. Sequence analysis of 22 independent mCAT-2 cDNA clones reveals that the cDNA sequences converge precisely 16 bp 5' of the initiator AUG codon. Moreover, analysis of genomic clones shows that the mCAT-2 gene 5'UTR exons are dispersed over 18 kb. Classical promoter and enhancer elements are present in appropriate positions 5' of the exons and their utilization results in regulated mCAT-2 mRNA accumulation in skeletal muscle and liver following partial hepatectomy. The isoform adjacent to the most distal promoter is found in all tissues and cell types previously shown to express mCAT-2, while the other 5' UTR isoforms are more tissue specific in their expression. Utilization of some or all of five putative promoters was documented in lymphoma cell clones, liver, and skeletal muscle. TATA-containing and (G+C)-rich TATA-less promoters appear to control mCAT-2 gene expression. The data indicate that the several distinct 5' mCAT-2 mRNA isoforms result from transcriptional initiation at distinct promoters and permit flexible transcriptional regulation of this cationic AA transporter gene.

Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.

We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower-level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity.

The broad gauge, the bane of the Great Western Railway Company : with an account of the present & prospective liabilities saddled on the proprietors by the promoters of that peculiar crotchet /

Mode of access: Internet.

Appeal to members of Parliament, and to the working classes, respecting the ten hours factory bill : with sketches of its chief promoters /

Signed: Philo Demos.

Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and in a subset of tissue macrophages and dendritic cells. It is expressed at lower levels in the parenchymal cells of the liver, glomerular mesangial cells of the kidney and pancreatic acinar cells. We have identified novel TRAP mRNAs that differ in their 5-untranslated region (5'-UTR) sequence, but align with the known murine TRAP mRNA from the first base of Exon 2. The novel 5'-UTRs represent alternative first exons located upstream of the known 5'-UTR. A similar genomic structure exists for the human TRAP gene with partial conservation of the exon and promoter sequences. Expression of the most distal 5'-UTR (Exon 1A) is restricted to adult bone and spleen tissue. Exon 1B is expressed primarily in tissues containing TRAP-positive nonhaematopoietic cells. The known TRAP 5'-UTR (Exon 1) is expressed in tissues characteristic of myeloid cell expression. In addition the Exon 1C promoter sequence is shown to comprise distinct transcription start regions, with an osteoclast-specific transcription initiation site identified downstream of a TATA-like element. Macrophages are shown to initiate transcription of the Exon 1C transcript from a purine-rich region located upstream of the osteoclast-specific transcription start point. The distinct expression patterns for each of the TRAP 5'-UTRs suggest that TRAP mRNA expression is regulated by the use of four alternative tissue- and cell-restricted promoters. (C) 2003 Elsevier Science B.V. All rights reserved.

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome.

Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NATI (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NATI is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5'non-coding region of NATI. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NATI expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforrns were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NATI transcripts may contribute to the variation in NATI activity in vivo.