Both TATA box and upstream regions are required for the nopaline synthase promoter activity in transformed tobacco cells

Summary Using a promoter expression vector system based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, we have studied the molecular structure of the nopaline synthase (nos) promoter which is active constitutively in transformed plant tissues. The system uses the sensitive and reliable chloramphenicol acetyltransferase (CAT) assay for the analysis of promoter strength in plant cells. Two sets of mutants were generated by sequential deletion of the nos promoter region from both 5 and 3 ends. These promoter fragments were linked to the cat coding sequence within the expression vector. The strength of the mutant promoters was measured in transformed tobacco calli as CAT activity. 3 deletions up to-17 bp did not significantly affect the promoter strength. Further deletions into the TATA box region reduced the promoter strength by about ten-fold. Analysis of the 5 deletion mutants showed that an upstream region is required for the nos promoter activity in addition to the TATA box and CCAAT box regions.     

Sequence-specific interactions of wound-inducible nuclear factors with mannopine synthase 2′ promoter wound-responsive elements

A 318 bp mannopine synthase 2 (mas2) promoter element from the T-DNA of Agrobacterium tumefaciens can direct wound-inducible and root-preferential expression of a linked uidA gene in transgenic tobacco plants. Wound inducibility is further enhanced by sucrose in the medium. Promoter deletion analysis indicated that the sucrose enhancement is conferred by a region extending from –318 to –213. DNase I footprinting indicated that an A/T-rich DNA sequence in this region is protected by tobacco nuclear factors. Regions extending from –103 to +66 and from –213 to –138 directed wound-inducibile expression of a linked uidA gene when placed downstream of a CaMV 35S enhancer or upstream of a truncated (–209) CaMV 35S promoter, respectively. DNase I footprinting analyses indicated that proteins from wounded tobacco leaves specifically bound to three contiguous motifs downstream of the mas2 TATA box. In addition to a common retarded band formed by the upstream wound-responsive element complexed with proteins from either wounded or unwounded tobacco leaves, two unique retarded bands were observed when this element was incubated with protein from wounded leaves. Methylation interference analysis additionally identified an unique motif composed of promoter elements and nuclear factors derived specifically from wounded tobacco leaves. We propose a model to describe the involvement of nuclear factors with mas2 promoter elements in wound-inducible gene expression.     

DNA variants within the 5′-flanking region of milk-protein-encoding genes II. The β-lactoglobulin-encoding gene

For the detection of polymorphisms within the 5-flanking region of the -lactoglobulin (-LG) -encoding gene a nucleotide sequence containing 795 bp of the promoter and 59 bp of exon I was cloned and sequenced. After comparing the sequence from the DNA of 11 diverse cows (different breeds and milk-protein yields), 14 singlebp substitutions were identified within the 5-flanking region and two in the 5-untranslated region (5-UTR) of exon I. Some of the variants are located in potential binding sites for trans-acting factors or in the 5-UTR. A PCR-based RFLP analysis was performed, and the genotypes of an additional 60 cows were identified at five variable 5-flanking sites. The results reveal three frequent combinations between the A and B alleles of the protein-coding region and the novel 5-flanking DNA variants. This finding may explain the differences of the protein-variant-dependent -LG synthesis (A>B) observed in vivo. A sequence comparison of the bovine and ovine promoters reveals an homology of 92.8% and shows a higher degree of conservation between positions -600 and -300.     

Characteristics of a strong promoter from figwort mosaic virus: comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter

A segment of DNA from the genome of figwort mosaic virus (FMV) strain M3 possesses promoter activity when tested in electroporated protoplasts from, and transgenic plants of, Nicotiana tabacum cv. Xanthi nc. The 1.1 kb DNA segment, designated the 34S promoter, is derived from a position on the FMV genome comparable to the position on the cauliflower mosaic virus (CaMV) genome containing the 35S promoter. The 34S and 35S promoters show approximately 63% nucleotide homology in the TATA, CCACT, and –18 to +1 domains, but in sequences further upstream the homology drops below 50%. Promoter activities were estimated using -glucuronidase and neomycin phosphotransferase II reporter gene systems. The activity of the 34S promoter segment approximates that of the 35S promoter in both protoplast transient expression assays and in stably transformed tobacco plants. Truncation of 5 sequences from the 34S promoter indicates that promoter strength depends upon DNA sequences located several hundred nucleotides upstream from the TATA box. In leaf tissue the 34S promoter is 20-fold more active than the mannopine synthase (MAS) promoter from Agrobacterium tumefaciens T-DNA. The 34S promoter lacks the root-specific and wound-stimulated expression of the MAS promoter, showing relatively uniform root, stem, leaf, and floral activities.     

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter

A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5 to 3) in the 5 region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter--glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position-173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the-326 to-173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position-173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.     

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1α is conserved among angiosperms

In Arabidopsis thaliana, the activation process of the A1 EF-1 gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5 non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5 intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.     

Isolation of an autonomously replicating sequence (ARS) from satellite DNA of Drosophila melanogaster

Summary Autonomously replicating sequences (ARSs) were cloned from the 1.688 satellite DNA of D. melanogaster using YIp5, consisting of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^– yeast strain as the recipient. Three out of six clones contained an ARS and the average frequncy of the occurrence of ARS was thus calculated to be approximately one per 14 kbp of the satellite DNA. A 500 bp ARS fragment (BgHS500) was obtained from one of the resultant clones (pYDS57). BgHS500 does not hybridize with the major repeating unit (370 bp) but it does with the minor unique sequence of the satellite. The sequence of BgHS500 was determined and found to be rich in AT and to contain the sequence, 5AAAACATAAAA3, a sequence common to yeast ARSs. However, a smaller fragment (150 bp) isolated from BgHS500 and containing the 11 bp sequence did not exhibit the characteristics of an ARS. The average copy number in the transformants of pBgHS500, a recombinant molecule of BgHS500 and YIp5, ranged from 0.05–0.5, while that of the parent plasmid, pYDS57, was about 2–10. On the basis of these results, it is postulated that the sequence 5AAAACATAAAA3 may possibly consiitute the core of ARSs and certain other sequences may also be necessary to insure that the ARS consistently undergoes at least one complete replication in each cell cycle. The role of ARSs in the genome of D. melanogaster is discussed.     

Upstream non-coding region which confers polar expression to Ri plasmid root inducing gene rolB

Summary Root differentiation could be elicited on carrot discs by transformation with the agropine Ri plasmid rolB gene cloned in the binary vector Bin19, provided two conditions were met. Firstly, an adequate auxin supply had to be provided. This was achieved by co-inoculation with a strain carrying only the auxin synthetic genes of the T_R-DNA. Most of the resulting roots were then shown to harbour only rolB and no aux genes. Secondly, an extended non-coding region (1200 bp) at the 5 end of rolB had to be included in the construction. A shorter (300 bp) 5 region, including TATA and CCAAT boxes, was not sufficient to trigger root differentiation. Both the extended (B1185) and reduced (B310) 5 regions of rolB were then cloned upstream of the -glucuronidase (GUS) reporter gene and infections carried out both on the apical and on the basal side of carrot discs. Strong expression of GUS, visualized histochemically as an intense blue colouring of transformed cells was observed with B1185-GUS constructions on the apical side of the discs. Only occasionally could coloured cells be observed on the basal side of the discs with B1185-GUS and on both apical and basal sides with B310-GUS constructions. Strong GUS expression was, on the contrary, achieved on cells of both auxin-rich (apical) and auxin-depleted (basal) sides of the discs with the strong constitutive viral promoter, CaMV35S. These results indicate the presence of an upstream regulatory region which confers polar expression to the rolB gene and suggest a role for auxin in its activation.     

Regulated spatial expression of fusion gene constructs with the 5′ upstream region of Halocynthia roretzi muscle actin gene in Ciona savignyi embryos

pHrMA4a-Z is a recombinant plasmid in which about 1.4 kb of the 5 flanking region of a gene for muscle actin HrMA4a from the ascidian Halocynthia roretzi is fused with the coding sequence of a bacterial gene for -galactosidase (lac-Z). In this study, we examined the expression of the fusion gene construct when it was introduced into eggs of another ascidian, namely Ciona savignyi. When a moderate amount of linearized pHrMA4a-Z was introduced into fertilized Ciona eggs, the expression of the reporter gene was evident in muscle cells of the larvae, suggesting that both species share a common machinery for the expression of muscle actin genes. The 5 upstream region of HrMA4a contains several consensus sequences, including a TATA box at -30, a CArG box at -116 and four E-boxes within a region of 200 bp. A deletion construct, in which only the 216-bp 5 flanking region of HrMA4a was fused with lac-Z, was expressed primarily in larval muscle cells. However, another deletion construct consisting of only the 61-bp upstream region of HrMA4a fused with lac-Z was not expressed at all. When pHrMA4a-Z or pHrMA4a-Z (–216) was injected into each of the muscle-precursor blastomeres of the 8-cell embryo, expression of the reporter gene was observed in larval muscle cells in a lineage-specific fashion. However, expression of the reporter gene was not observed when the plasmid was injected into non-muscle lineage. Therefore, the expression of the reporter gene may depend on some difference in cytoplasmic constituents between blastomeres of muscle and non-muscle lineage in the 8-cell embyo.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.     

Molecular characterization of two clusters of genes encoding the Type I CAB polypeptides of PSII in Nicotiana plumbaginifolia

A Nicotiana plumbaginifolia genomic library in the phage Charon 34 was used to isolate and characterize 7 full-length genes and part of an 8th gene encoding chlorophyll a/b-binding (CAB) polypeptides. These genes are arranged in two clusters. All the genes within the clusters are arranged in opposite orientation to their neighbours. The nucleotide sequences of two genes, one from each cluster, show that both genes, designated Cab-E and Cab-C, encode very similar proteins (95.9% of homology) corresponding to type I photosystem II polypeptides. Southern blot analysis suggests that at least 19 CAB genes encoding type I PSII CAB polypeptides are present in the N. plumbaginifolia genome. We also describe the presence within the N. plumbaginifolia genome of CAB genes encoding PSII type II CAB polypeptides and PSI type I CAB polypeptides. The sequences of the 5 flanking region of three different CAB genes (Cab-E, Cab-C, and CAB-F) were determined. Two of them (Cab-C and Cab-F) share extensive homology, whereas the Cab-E promoter shows homology to Cab-C and Cab-F only in a unique region extending from the CAAT box to the TATA box. This conserved sequence is also found in the same position in promoters of CAB genes encoding type I PSII polypeptides from other plant species.Abbreviations CAB chlorophyll a/b-binding protein - bp base pair(s) - kb 1 000 bp     

Upstream sequences regulating legumin gene expression in heterologous transgenic plants

Summary We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the -glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. -Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.     

Sugar response element enhances wound response of potato proteinase inhibitor II promoter in transgenic tobacco

The promoter region of the potato proteinase inhibitor II (PI-II) gene was studied to identifycis-acting regulatory sequences involved in sugar response using transgenic tobacco plants. The 5 control region covering an 892 nucleotide sequence upstream from the cap site and a 32 nucleotide untranslated region of the PI-II promoter was able to activate a reporter chloramphenicol acetyltransferase (cat) gene by wounding or by incubating in a sugar-free medium. This wound response was further enhanced by sugar. Hexoses, disaccharides, and some trisaccharides were strong inducers whereas pentoses, deoxy sugars, sugar acids, TCA cycle intermediates, amino acids, and other carbohydrates had little effect on the promoter activity. Deletion of the sequence between-892 and-573 abolished the wound response but not the sugar response. An additional 5 deletion to-453 removed the sugar inducibility. Locations of thecis-acting regulatory elements were further elucidated by 3 deletion analysis. Deletion of the downstream region from-520 did not affect the wound of sugar response of the promoter. However, 3 deletion mutant-574 was unable to respond to sugar but did respond weakly to wounding. Further deletion to-624 abolished both responses. Therefore, it can be concluded that a wound response element is located in between-624 and-574 and that the response is further enhanced by a sugar response element located in the sequence between-573 and-520.     

A proximal upstream sequence controls tissue-specific expression of <Emphasis Type="Italic">Lem2</Emphasis>, a salicylate-inducible barley lectin-like gene

The lemma and palea (lemma/palea), which form the husk of barley (Hordeum vulgare L.) seeds, constitutively express high levels of defense-related genes, relative to leaves [Abebe et al. (2004) Crop Sci 44:942–950]. One of these genes, Lem2, is expressed mainly in the lemma/palea and coleoptile and is strongly upregulated by salicylic acid (SA) and its functional analog 2,6-dichloroisonicotinic acid . Induction by SA was rapid, occurring within 4 h of treatment. However, Lem2 is not responsive to methyl jasmonate (MeJA) or wounding and is downregulated by drought, dehydration, and abscisic acid. These results suggest that Lem2 is involved in systemic acquired resistance. Sequence analysis showed that LEM2 is a jacalin-related lectin (JRL)-like protein with two domains. Consistent with northern and western blot data, transient expression analyses using Lem2::gfp constructs showed strong expression in lemmas and a trace expression in leaves. Successive 5 deletions of the 1,414 bp upstream region gradually weakened promoter strength, as measured by real-time PCR. Promoter deletion studies also revealed that the –75/+70 region (containing the TATA box, 5 UTR, and a SA-response element) determines tissue specificity and that the distal promoter region simply enhances expression. Southern analysis indicated that Morex barley has at least three copies of the Lem2 gene arranged in tandem on chromosome 5(1H) Bin 02, near the short arm telomere. Lem2 is not present in the barley cultivars Steptoe, Harrington, Golden Promise, and Q21861.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture     

Kidney-Specific Activity of the Bovine Uromodulin Promoter

A 10-kilobase (kb) bacteriophage bovine genomic clone containing 5.4 kb of the 5-flanking region, exons, and introns of bovine uromodulin gene was isolated. Transgenic mice containing 3.9 kb of the bovine uromodulin promoter and a lacZ reporter gene were generated by pronuclear microinjection. RT-PCR and northern blot analyses of transgene expression in various tissues of founder and F1 mice showed that the transgene was expressed exclusively in the kidney. In situ hybridization and histochemistry for lacZ demonstrated that transgene expression was restricted to tubule epithelial cells of the loop of Henle in the kidney. Stepwise 5 deletion analysis revealed that transfection of luciferase reporter constructs fused to various proximal 5-flanking regions of the bovine uromodulin gene markedly increased luciferase activity in mouse renal epithelial cells but not in mesenchymal cells and that the most critical cis elements of the uromodulin gene are located within the 600 bp upstream region.     

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of β-glucuronidase in transgenic Brassica plants

To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene -glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.     

Regulation of plant genes specifically induced in nitrogen-fixing nodules: role of cis-acting elements and trans-acting factors in leghemoglobin gene expression

Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7: 1265–1271, 1988). Subfragments of the S. rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181–191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100°C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srglb3 5 upstream region were fused to the -glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters. These constructs were used to generate transgenic Lotus corniculatus plants and their expression was measured in different plant tissues. The Srglb3 CAAT and TATA box region was found to be required for nodule-specific expression and several upstream enhancer-type regions were identified.     

Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.)

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev