Light-regulated expression of the pea plastocyanin gene is mediated by elements within the transcribed region of the gene

Expression of the pea plastocyanin gene ( PetE ) is regulated by light in both pea and transgenic tobacco plants. However, the PetE promoter with the 5' untranslated leader region does not direct light-regulated expression of the GUS reporter gene in transgenic tobacco. This suggested that sequences downstream of the translation start of the PetE gene are required for light-regulated expression. To investigate this possibility the expression of a series of chimeric gene constructs in transgenic tobacco plants was examined to assess the contributions of the promoter, the 5' untranslated leader region, the coding region and the 3' region of the PetE gene to light-regulated expression. Both the coding region and the 5' untranslated leader region of the PetE gene were found to be required for full light regulation. Full light regulation of chimeric gene constructs containing the cauliflower mosaic virus (CaMV) 35S promoter required the deletion of CaMV 5' leader and polylinker sequences from the constructs. The presence of CaMV and polylinker sequences at the 5' end of the PetE leader masked the light regulation directed by the transcribed region of the pea PetE gene.     

A two-component nodule-specific enhancer in the soybean N23 gene promoter.

The two positive cis elements in the soybean nodulin N23 gene promoter were investigated in transgenic Lotus corniculatus plants and shown to constitute a two-component nodule-specific enhancer. Equal quantitative contributions from the two components were suggested by the similar expression level of chimeric N23-chloramphenicol acetyltransferase genes after deletion of either the distal positive element (PE-A, -320 to -298) or the proximal positive element (PE-B, -257 to -165). A combined effect of the two elements was indicated by orientation-dependent effects in the N23 promoter, and by the observation that neither PE-A nor PE-B separately was able to confer any activity to the cauliflower mosaic virus 35S minimal promoter. Reactivation of the minimal N23 and the minimal cauliflower mosaic virus 35S promoters by the inverted complete element (PE-AB) further suggested that PE-AB is a nodule-specific enhancer containing two equally strong enhancer components. Two 12-bp sequence motifs, InvA and InvB, constituting an inverted repeat, were identified as the core of the enhancer components PE-A and PE-B, respectively. Point mutations in InvA or InvB resulted in lower expression levels and mutations in both abolished enhancer activity. Point mutations in two nodulin consensus sequences, 5'-CTCTT and 5'-AAAGAT located downstream of PE-AB, resulted in a decreased level of expression, confirming the involvement of these two motifs in nodulin gene expression. The binding site for the nodule-specific trans-acting factor, NAT2, present in the PE-A segment, was removed without affecting expression significantly. This interaction is, therefore, dispensable for enhancer activity.     

Analysis of rice Act1 5' region activity in transgenic rice plants.

The 5' region of the rice actin 1 gene (Act1) has been developed as an efficient regulator of foreign gene expression in transgenic rice plants. To determine the pattern and level of rice Act1 5' region activity, transgenic rice plants containing the Act1 5' region fused to a bacterial beta-glucuronidase (Gus) coding sequence were generated. Two independent clonal lines of transgenic rice plants were analyzed in detail. Quantitative analysis showed that tissue from these transgenic rice plants have a level of GUS protein that represents as much as 3% of total soluble protein. We were able to demonstrate that Act1-Gus gene expression is constitutive throughout the sporophytic and gametophytic tissues of these transgenic rice plants. Plants from one transgenic line were analyzed for the segregation of GUS activity in pollen by in situ histochemical staining, and the inheritance and stability of Act1-Gus expression were assayed in subsequently derived progeny plants.     

Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana

The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes (sam). Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene. Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue. This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes. Using a chimeric beta-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A. thaliana plants. A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf. However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter.     

Expression of. Arabidopsis tryptophan biosynthetic pathway genes: effect of the 5' coding region of phosphoribosylanthranilate isomerase gene

There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5' region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5' coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5' coding region of PAI1 or PAI3 was 60—100-fold higher than that without the corresponding 5' region. However, the effect of 5' coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by t     

The developmental regulation of the human zeta-globin gene in transgenic mice employing beta-galactosidase as a reporter gene.

We have investigated the developmental and tissue specific expression of the human embryonic zeta-globin gene in transgenic mice. A construct containing 550 bp of zeta-globin 5' flanking region, fused to a beta-galactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like alpha positive regulatory element (alpha PRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire zeta-globin gene linked to the alpha PRE or the beta LCR. Data showed that 12% of mice generated from eggs injected with zeta-promoter/lacZ/alpha PRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire zeta-globin gene linked to the alpha PRE or the beta LCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, possibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that zeta-globin promoter activity was most pronounced at 8.5-9.5 days of development and was restricted to erythroid cells. By 15 days of development, no zeta-globin promoter activity was detected. These results suggest that the alpha PRE can direct high level expression from the zeta-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human zeta-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the zeta-globin gene or 3' of the cap site do not appear to be necessary for correct zeta-globin developmental regulation.     

A potato Sus3 sucrose synthase gene contains a context-dependent 3' element and a leader intron with both positive and negative tissue-specific effects.

To examine which sequences are involved in regulating the potato sucrose synthase gene Sus3-65, we examined a series of deletion and substitution constructs in transgenic potato and tobacco plants. In a construct containing 3.9 kb of 5' flanking region, substitution of the native 3' sequence with the nopaline synthase 3' sequence and deletion of the leader intron did not significantly affect expression in vegetative tissues. However, in a construct containing only 320 bp of 5' flanking region, these changes had marked effects. Replacing the native 3' sequences with nopaline synthase 3' sequences caused a six- to 20-fold increase in expression in vascular tissue, and removing the leader intron almost completely abolished expression in potato plants. Surprisingly, removal of the leader intron from either the full-length construct or a construct containing only 320 bp of 5' flanking sequence reduced expression in vascular tissue of tobacco anthers at later stages of development but increased expression in pollen by more than 100-fold.     

Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants.

The gln-gamma gene, which specifies the gamma subunit of glutamine synthetase in Phaseolus vulgaris L., has been isolated and the regulatory properties of its promoter region analyzed in transgenic Lotus corniculatus plants. A 2-kilobase fragment from the 5'-flanking region of gln-gamma conferred a strongly nodule-enhanced pattern of expression on the beta-glucuronidase reporter gene. Parallel studies on the promoter of another glutamine synthetase gene (gln-beta) showed that a 1.7-kilobase fragment directed 20-fold to 140-fold higher levels of beta-glucuronidase expression in roots than in shoots. Histochemical localization of beta-glucuronidase activity in nodules of the transgenic plants indicated that the chimeric gln-gamma gene was expressed specifically in the rhizobially infected cells; expression of the gln-beta construct was detected in both cortical and infected regions of young nodules, and became restricted to the vascular tissue as the nodule matured. We conclude that gln-beta and gln-gamma genes are differentially expressed both temporally and spatially in plant development and that the cis-acting regulatory elements responsible for conferring these contrasting expression patterns are located within a 2-kilobase region upstream of their coding sequences.     

Estrogen-inducible and liver-specific expression of the chicken Very Low Density Apolipoprotein II gene locus in transgenic mice.

We have examined the chicken Very Low Density Apolipoprotein II (apoVLDL II) gene locus in transgenic mice. A DNA fragment composed of the transcribed region, 16 kb of 5' flanking and 400 bp of 3' flanking sequences contained all the information sufficient for estrogen-inducible, liver-specific expression of the apoVLDL II gene. The far-upstream region contains a Negative Regulating Element coinciding with a DNaseI-hypersensitive site at -11 kb. In transgenic mice, the NRE at -11 kb is used for downregulating the expression to a lower maximum level. The NRE might be used for modulating apoVLDL II gene expression, and may be involved in the rapid shut-down of the expression after hormone removal.     

Deletion analysis of the human CD2 gene locus control region in transgenic mice.

Genomic sequences located at the 3' flanking region of the human CD2 gene confer high level tissue-specific, position-independent expression of the gene when introduced in the germ line of mice. In order to further characterize these sequences a range of deletions, from the 3' end were produced and transgenic mice were generated with the human CD2 (hCD2) gene linked to these deleted fragments. This allowed us to establish the minimum sequences necessary for the copy-dependent transgene expression. 2.1 kb or 1.5 kb of 3' flanking sequences linked to a hCD2 mini-gene is sufficient to allow T-cell specific, copy-dependent, integration-independent expression in transgenic mice. 1.1 kb of 3' sequences results in the gene being expressed in a T-cell specific manner, but copy-dependent, integration-independent expression was not observed in a small number of transgenic animals. 0.2 or 0.5 kb of 3' flanking sequences were insufficient to allow expression above the level previously found with a human CD2 gene which lacked 3' flanking sequences. We conclude that the Locus Control Region (LCR) effect is caused by 1.5 kb of flanking sequences immediately 3' to the polyadenylation signal of the gene.