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PubMed1996

Analysis of kafirin promoter activity in transgenic tobacco seeds.

Sequences corresponding to 855 bp of 5' promoter region and the transit peptide from lambdaGK.1,a genomic clone encoding a 22 kDa alpha-kafirin seed protein from sorghum, were translationally fused to a cloned beta-glucuronidase (GUS) coding sequence from uidA and transferred to tobacco via Agrobacterium tumefaciens-mediated transformation. No GUS expression was detectable at any stage of growth in stems or leaves of these plants. However, GUS expression was detected in both embryo and endosperm tissues of resulting tobacco seeds 10-15 days after flowering. Dissected tissues indicate endosperm expression was localized within the bulk endosperm and not within the parenchyma cell layer underlying the integument. These studies also demonstrate that within dissected tobacco embryos, expression from the kafirin promoter was restricted to the mesocotyl region.

Cloning, Molecular↗

PubMed1996

Promoter elements controlling developmental and environmental regulation of a tobacco ribosomal protein gene L34.

The rpL34 gene, which encodes a cytoplasmic ribosomal protein with a high homology to the rat 60S r-protein L34, was isolated from a genomic library of tobacco (Nicotiana tabacum L. cv. Xanthi-nc). A 1500 bp upstream promoter fragment was fused to the chloramphenicol acetyltransferase (CAT) reporter gene or beta-glucuronidase (GUS) reporter gene and transferred into tobacco plants by the Agrobhacterium-mediated leaf disk transformation method. Analysis of CAT activity in leaf tissues showed that mechanical wounding increased the rpL34 promoter activity about 5 times as compared to untreated controls and that the promoter activity was further enhanced by plant growth regulators, 2,4-dichlorophenoxyacetic acid and benzyladenine. Histochemical GUS staining patterns of the transgenic plants showed that the rpL34 promoter activity is high in actively growing tissues, including various meristems, floral organs, and developing fruits. A series of 5' deletion analyses of the rpL34 promoter indicated that a 50 bp region located between -179 and -129 is essential for wound, auxin and cytokinin responses. Deletion of this region reduced the promoter activity to an undetectable level. Insertion of the 50 nucleotide sequence into a minimal promoter restored the promoter activity and the promoter strength was proportional to the copy number of the upstream sequence. The role of TATA and CAAT box regions was studied by a series of 3' deletion analyses. A 3' deletion up to -28 did not significantly affect the promoter strength. However deletion of the promoter up to 70 bp, which deleted the TATA box region, significantly reduced promoter activity. Further deletion of the promoter up to - 104. eliminating the CAAT box region, abolished the promoter activity. These results suggest that the TATA box and CAAT box regions are also important for the rpL34 promoter activity in addition to the 50 bp upstream region.

Base Sequence↗

PubMed1995

Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plants.

The heat shock (hs) response during plant growth and development was analyzed in tobacco and Arabidopsis using chimaeric beta-glucuronidase reporter genes (hs-Gus) driven by a soybean hs promoter. Fluorimetric measurements and histochemical staining revealed high Gus activities in leaves, roots, and flowers exclusively after heat stress. The highest levels of heat-inducible expression were found in the vascular tissues. Without heat stress, a developmental induction of hs-Gus was indicated by the accumulation of high levels of Gus in transgenic tobacco seeds. There was no developmental induction of hs-Gus in Arabidopsis seeds. In situ hybridization to the RNA of the small heat shock protein gene Athsp17.6 in tissue sections revealed an expression in heat-shocked leaves but no expression in control leaves of Arabidopsis. However, a high level of constitutive expression of hs genes was detected in meristematic and provascular tissues of the Arabidopsis embryo. The developmental and tissue-specific regulation of the hs response is discussed.

Arabidopsis↗

PubMed1995

Expression of Agrobacterium rhizogenes auxin biosynthesis genes in transgenic tobacco plants.

Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes. Neither wound nor hormone induction could be detected on transgenic leaf discs. However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts. The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli. Histological analysis showed that the expression was located in cells reactivated by in vitro culture. Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the beta-glucuronidase activity of the chimaeric genes. These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.

Cell Cycle↗

PubMed1995

Structural and functional characterization of two wheat histone H2B promoters.

Two wheat histone H2B genes (TH123 and TH153) were isolated. Nucleotide sequence analysis revealed that some characteristic sequence motifs were conserved in both the 5'- and 3'-flanking regions. A canonical TATA box and several CCAAT sequences were present in the presumed promoter regions. Motifs similar or identical to the hexamer (ACGTCA) and octamer (CGCGGATC) motifs that are positive cis-acting elements of the wheat H3 (TH012) promoter were also observed in both the H2B promoters. A gel mobility shift assay indicated that the hexamer and hexamer-like motifs bound the wheat bZIP proteins HBP-1a and/or HBP-1b in vitro. A novel sequence motif, (A/T)(G/A)AAAT(A/G), was found downstream of a translational stop codon as observed in several plant histone H2B cDNAs. Promoter activity was analyzed with H2B promoter-GUS fusion genes in the transient system using tobacco protoplasts. Studies of the promoter function in transgenic tobacco plants showed that the H2B promoters were preferentially active in meristematic tissues. Taken together, our data indicate that the H2B genes are regulated, in part, by the same mechanism as found in H3 and H4 gene transcription.

Amino Acid Sequence↗

PubMed1995

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum.

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S2 clone, with 1.6 kb of 5' sequence, nor the S6 clone, with 2.8 kb of 5' sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S2 clone was not detected, however the S6 clone was expressed (at low levels) in three out of four transgenic plants. An S6-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.

Cloning, Molecular↗

PubMed1993

Localization of light-inducible and tissue-specific regions of the spinach ribulose bisphosphate carboxylase/oxygenase (rubisco) activase promoter in transgenic tobacco plants.

Deletions in the spinach rubisco activase (Rca) promoter in transgenic tobacco were analyzed to define the regions necessary for conferring light-inducible and tissue-specific expression. Transgenic plants were constructed with Bal 31 deletions of the Rca promoter fused to the coding region of the bacterial reporter gene beta-glucuronidase (GUS). Analysis of the Rca deletion mutants localized the region conferring normal expression downstream from -294 relative to the Rca transcription start site. A second set of transgenic plants containing the cauliflower mosaic virus (CaMV) 35S enhancer fused to the 3' end of the Rca/GUS constructs demonstrated the presence of a light-responsive element between -150 and -78 active in leaves. Regions 10 bp long within the light-responsive region, which included putative G box and GT elements, were removed by recombinant polymerase chain reaction. Deletion of the G box element resulted in a loss of gene expression in the leaves of transgenic tobacco, while deletion of the GT motif caused a 10-100-fold increase in expression in roots. However, site-directed mutagenesis of the GT motif resulted in expression patterns identical to the normal promoter. These experiments demonstrated that light-inducible and tissue-specific expression of the Rca promoter involves multiple cis elements proximal to the transcription start site, and that interactions between these elements are essential for regulating expression.

Gene Expression Regulation↗

PubMed1993

A poplar tree proteinase inhibitor-like gene promoter is responsive to wounding in transgenic tobacco.

Wounding of poplar trees leads to the accumulation of several mRNA species that encode proteins with putative defensive function. One class of wound-induced poplar RNA (win3) has amino acid sequence similarity to Kunitz-type trypsin inhibitors. Northern blots and cDNA sequencing show that several win3 mRNAs accumulate in the uninjured leaves of wounded trees. We report further characterization of the win3 family including sequence comparisons, gene family organization, and the identification of one win3 member that is transcriptionally activated in response to mechanical wounding. We also show that 1.5 kb of 5'-flanking sequence of one win3 member (win3.12) is sufficient to confer wound-regulated expression of a beta-D-glucuronidase (GUS) reporter gene in transgenic tobacco. Annual herbaceous plants such as tobacco can thus be used to study the expression of genes from a perennial woody angiosperm.

Amino Acid Sequence↗

PubMed1995

Expression of the auxin-regulated parA gene in transgenic tobacco and nuclear localization of its gene products.

An auxin-regulated gene, parA, comprises a gene family consisting of a handful genes which respond to various signals. Although Droog et al. (Plant Mol. Biol, 1993, 21, 965-972) postulated that the parA-related genes belong to the family of a cytoplasmic enzyme, glutathione S-transferase (GST), we detected a low level of GST activity in the parA products, whose value was below 1/30 of that of parB products encoding tobacco (Nicotiana tabacum L.) GST. Immunofluorescence studies using an antibody against parA protein revealed that the subcellular location of parA protein is the nucleus in cultured tobacco mesophyll protoplasts, while conventional GSTs' including the parB product were primarily located in the cytoplasm. Confocal laser scanning microscopy of tobacco BY-2 cells showed that the parA product was confined to the nucleus, but was excluded from the nucleolus. In addition, exon/intron organization of the parA family was appreciably different from that of conventional GSTs including parB. Furthermore, the parA protein is much more similar to a 24-kDa protein of Escherichia coli that is reported to bind to RNA polymerase. These different characteristics of parA compared with to the conventional GSTs, indicate that parA protein would have distinct functions, such as involvement in transcription, rather than functioning as a conventional GST. Transgenic tobacco plants that carried the parA promoter fused to a beta-glucuronidase gene were used to show that the parA gene is tissue-specific and also under developmental control.

Amino Acid Sequence↗

PubMed1995

Deletion analysis of the Brassica napus cruciferin gene cru 1 promoter in transformed tobacco: promoter activity during early and late stages of embryogenesis is influenced by cis-acting elements in partially separate regions.

To define sequences in the cruciferin gene cru1 promoter of importance for expression, tobacco (Nicotina tabacum L.) plants were transformed with constructs in which the cru1 promoter, in front of the intact cru1 structural gene, was truncated at -1216, -974, -736, -515, -306, -46 and -17 bp relative to the cap-site. Cru1 expression in tobacco seeds was studied by Northern analysis, Western analysis and in-situ hybridizations. Comparisons of the Northern analysis of RNA from tobacco seeds harvested at 18 d after pollination with the Western analysis of protein from mature seeds showed that the regions between -974 to -736 and -306 to -46 were important for the expression of cru1 at an early developmental stage, whereas the regions -736 to -515 and -515 to -306 were important for expression throughout embryogenesis. By investigating the mRNA levels in transgenic seeds at different stages of development, indications were obtained that the two latter regions exerted their effects during the later stages. The in-situ hybridization showed that cru1 mRNA was distributed in parenchyma cells throughout the embryo in seeds expressing constructs -974 and -736. Constructs -515 and -306 showed an expression restricted to the axis or axis and parts of the cotyledons. Sequence comparisons of the cru1 promoter with other storage-protein gene promoters, identified several motifs implicated in gene regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗

PubMed1993

Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants.

We characterized the expression of genes that correspond to a cDNA clone, RD29, which is induced by desiccation, cold and high-salt conditions in Arabidopsis thaliana. Northern analysis of desiccation-induced expression revealed a two-step induction process. Early induction occurs within 20 min and secondary induction occurs 3 h after the start of desiccation. Exogenous abscisic acid (ABA) induces RD29 mRNA within 3 h. Two genes corresponding to RD29, rd29A and rd29B, are located in tandem in an 8 kb region of the Arabidopsis genome and encode hydrophilic proteins. Desiccation induces rd29A mRNA with two-step kinetics, while rd29B is induced only 3 h after the start of desiccation. The expression of both genes is stimulated about 3 h after application of ABA. It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to desiccation and the other induced by changes in osmotic potential. The beta-glucuronidase (GUS) reporter gene driven by the rd29A promoter was induced at significant levels by desiccation, cold, high-salt conditions and ABA in both transgenic Arabidopsis and tobacco. Histochemical analysis of GUS activity revealed that the rd29A promoter functions in almost all the organs and tissues of vegetative plants during water deficiency.

Abscisic Acid↗

PubMed1994

Co-suppression of nitrate reductase host genes and transgenes in transgenic tobacco plants.

Constructs carrying the entire or part of the tobacco nitrate reductase cDNA (NIA) cloned between the promoter and terminator sequences of the 35S RNA of the cauliflower mosaic virus were introduced into tobacco, in an attempt to improve nitrate assimilation. Several transgenic plants that had elevated NIA mRNA and nitrate reductase (NR) activity were obtained. In addition, a few plants that exhibited a chlorotic phenotype characteristic of NR-deficient mutants were also obtained. One of these plants contained no NIA mRNA, no NR activity and accumulated nitrate. This phenotype was therefore assumed to result from co-suppression of 35S-NIA transgenes and host NIA genes. NR-deficient plants were also found among the progeny of transformants overexpressing NIA mRNA. Genetic analyses indicated that these NR-deficient plants were homozygous for the 35S-NIA transgene, although not all homozygous plants were deficient for NR. The ratio of normal to NR-deficient plants in the progeny of homozygous plants remained constant at each generation, irrespective of the state of expression of the NIA genes (active or inactive) in the previous generation. This ratio also remained unchanged when field trials were performed in two areas of France: Versailles and Bergerac. The analysis of homozygous plants revealed that co-suppression was reversible at some stage of sexual reproduction. Indeed, host genes and transgenes reactivated at each generation, and co-suppression always appeared after a lag period of normal growth, suggesting that the phenomenon is developmentally regulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Caulimovirus↗

PubMed1992

Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco.

The thermoregulated expression of the soybean heat shock (hs) gene Gmhsp17.3-B is regulated via the heat shock promoter elements (HSEs), but full promoter activity requires additional sequences located upstream of the HSE-containing region. Structural features within this putative enhancer region include a run of simple sequences which are also present upstream of HSE-like sequences of other soybean hs genes, and three perfect CCAAT box sequences located immediately upstream from the most distal HSE of the promoter. A series of heterologous and homologous promoter fusions linked to the chloramphenicol acetyl transferase (CAT) gene was constructed and examined in transgenic tobacco plants. The region containing the AT-rich domain of the 5' flanking region was unable to direct transcription from the TATA box of a truncated delta CaMV35S promoter. Heat-inducible CAT activity was detectable when additional sequences from the native promoter containing three CCAAT boxes and a single HSE were present in the constructions. Complete reconstitution of the native hs promoter/enhancer region increased hs specific CAT activities only very little, but deletion of CCAAT box sequences reduced CAT expression five-fold. Our results suggest that AT-rich sequences have a moderate effect on thermoinducible expression levels of the soybean heat shock gene and that CCAAT box sequences act cooperatively with HSEs to increase the hs promoter activity.

Base Sequence↗

PubMed1992

A plant histone gene promoter can direct both replication-dependent and -independent gene expression in transgenic plants.

Chimeric genes containing the beta-glucuronidase (GUS) gene under the control of different Arabidopsis histone H3 and H4 promoters were found to be highly expressed in transient expression experiments using tobacco protoplasts. The activity of one of these promoters, H4A748, was further analyzed. The kinetics of H4A748-GUS activity are very similar to these of a CaMV 35S-GUS constitutive gene during protoplast culture. No increase in H4A748-GUS activity was found after 24 h of protoplast culture when DNA synthesis starts, nor was the GUS activity affected when an inhibitor of DNA synthesis was included in the culture medium. This failure to detect any replication-dependent activity is most likely to be due to the fact that transient transcription of the introduced construct is restricted to the first 24 h following transfection. Stable integration of the H4A748-GUS gene into tobacco plants showed that the histone promoter could confer increased expression in meristematic tissues but it is also expressed to significant levels in non-proliferating tissues. Protoplasts prepared from these transgenic tobacco plants were cultivated under different conditions that affect DNA synthesis. Analysis of H4A748-GUS activity revealed (i) the existence of a basal replication-independent activity and (ii) a replication-dependent activity induced in parallel with DNA synthesis. These results show that the histone H4 promoter is able to direct both replication-dependent and -independent gene expression.

DNA↗

PubMed1994

The rolC promoter of Agrobacterium rhizogenes Ri plasmid is activated by sucrose in transgenic tobacco plants.

The 5'-upstream region of the rolC gene of the Ri plasmid is expressed specifically in phloem cells of transgenic higher plants. In this study, we demonstrated that the rolC promoter is activated by sucrose in phloem cells of transgenic tobacco seedlings bearing rolC promoter-uidA chimeric fusion gene. Since the rolC promoter is not activated by sorbitol, sucrose metabolism rather than osmotic pressure exerted by the disaccharide may be responsible for induction. Thus, experiments using 5'-upstream deletion mutants, internal deletion mutants, and chimeric constructs with a heterologous promoter (-90 region of the cauliflower mosaic virus 35S promoter) were conducted to define the region of the rolC promoter involved in sucrose activation. The results indicated that a cis-acting sucrose responsive region of the rolC promoter is located between -135 and -94 bp with respect to the transcription initiation site. In phloem cells, high concentrations of sucrose are encountered owing to ongoing translocation of photosynthates from source to sink tissues. Therefore, sucrose as a signal molecule may regulate the phloem-specific expression of the rolC promoter.

Bacterial Proteins↗

PubMed1993

Characterization of the promoter from the single-copy gene encoding ferredoxin-NADP(+)-oxidoreductase from spinach.

We describe a genomic DNA segment from spinach that bears part of the single-copy gene for ferredoxin-NADP(+)-oxidoreductase (FNR) including a 3.4 kb promoter sequence. Dissection of this DNA segment and its analysis in GUS (beta-glucuronidase) gene fusions in transgenic tobacco demonstrated that the promoter differs in structure from all other promoters for thylakoid protein genes studied to date. Two regions with light-responsive elements were identified. One is located within the first 118 bp upstream of the transcription initiation site. A second fragment covering nucleotide positions -220 to -119 is capable of conferring light-dependent GUS gene expression on two different minimal promoters. The latter fragment binds a transacting factor in gel-shift assays. None of the fragments carries cis elements known from other genes to be involved in light-controlled expression. Comparison of the light responsiveness of GUS gene fusions controlled by the -753/+231 and -118/+231 regions indicates that they respond differentially to phytochrome-dependent signals and that their expression in tobacco is not restricted to tissue with functional chloroplasts.

Amino Acid Sequence↗

PubMed1994

Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes.

Previous work has shown that two homologous, unlinked transgene loci can interact in plant nuclei, leading to non-reciprocal trans-inactivation and methylation of genes at one locus. Here, we report the structure and methylation of different transgene loci that contain the same construct but are variably able to inactivate and methylate a partially homologous, unlinked target locus. Silencing loci comprised multiple, methylated copies of the transgene construct, whereas a non-silencing locus contained a single, unmethylated copy. The correspondence between strength of silencing activity and copy number/degree of methylation was further demonstrated by producing novel alleles of a strong silencing locus: reducing the transgene copy number and methylation within this silencing locus decreased its ability to inactivate the target locus. The strong silencing locus, which was located close to a telomere, trans-inactivated various structural variants of the original target construct, regardless of their location in the genome. This suggests that the silencing locus can scan the entire genome for homologous regions, a process possibly aided by its telomeric location. Our data support the idea that epistatic trans-inactivation of unlinked, homologous transgenes in plants results from a pre-existing epigenetic difference between transgene loci, which is subsequently equalized by "epigene conversion" involving DNA-DNA pairing.

Alleles↗

PubMed1994

Susceptibility of transgene loci to homology-dependent gene silencing.

Previous work has shown that two unlinked, partially homologous transgene loci can interact in plant nuclei, leading to reversible methylation and inactivation of one transgene locus in the presence of the second. To study whether the chromosomal location of a transgene influences its susceptibility to trans-inactivation, we retransformed four transgenic lines, which contained the same construct (H) integrated in different chromosomal locations, with a second, partially homologous construct (K). At least 50 double transformants (DTs) were regenerated from each single transformants (ST) and screened for inactivation of markers [chloramphenicol acetyltransferase (CAT); hygromycin resistance (HYGR)] at the resident H locus. For two STs, H locus markers were inactivated in less than 1% of the DTs, suggesting that, at these integration sites, H was relatively resistant to trans-inactivation. In contrast, the other two STs appeared to be more sensitive to trans-inactivation: 4-10% of the DTs were CAT- and/or Hygs. Inactivation of H locus markers could be attributed to two distinct phenomena: 1. Regeneration from cells containing different epigenetic states of H, in which either both, one or none of the H alleles was active. This instability in the expression of the H locus, which was independent of K, was more pronounced in the homozygous state, and was associated with cellular mosaicism of expression and methylation. 2. The presence of an unlinked K locus could weaken the HygR phenotype by transcriptional inactivation and increased methylation of the hph gene at the H locus. These results indicated that a susceptible transgene locus is inherently unstable and partially methylated, and that these characteristics are exacerbated when the locus is homozygous for the transgene and/or when an unlinked homologous transgene is present.

Amino Acid Oxidoreductases↗