Spatial patterns of gene expression in Brassica napus seedlings: identification of a cortex-specific gene and localization of mRNAs encoding isocitrate lyase and a polypeptide homologous to proteinases.

We investigated the spatial expression of three genes that are expressed during seed germination and postgerminative development in Brassica napus L. using in situ hybridization procedures. Two of the mRNAs encode isocitrate lyase and a predicted polypeptide that is homologous to cysteine proteinases. We reported previously that the mRNAs are prevalent primarily in cotyledons of seedlings and accumulate with similar kinetics during postgerminative growth. Here, we show that the two mRNAs are detected in several seedling tissues, but they display different distribution patterns in both cotyledons and root-shoot axes. The third mRNA is abundant in seedling axes and accumulates specifically in the ground meristem and mature cortex of hypocotyls and roots. Distribution of the mRNA in root meristems suggests that the gene product participates in an early event in cortical cell differentiation. Our results provide insight into the physiological processes that characterize seedlings.     

Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37° C

We have analyzed the structure of genes encoding the glyoxylate cycle enzyme isocitrate lyase from Brassica napus L. and their expression during embryogeny and postgermination. Restriction mapping, nucleotide sequence, and DNA gel blot hybridization analyses of cDNA and genomic clones indicated that there are approximately six isocitrate lyase genes in the B. napus genome that can be divided into at least two subfamilies based upon their divergence in 5′ and 3′ untranslated regions. We showed previously that isocitrate lyase mRNA accumulates during late embryogeny and postgermination. Here, we present results which indicate that several isocitrate lyase genes are expressed at both stages of development. First, gene-specific probes were used to show that mRNAs encoded by representatives of both gene subfamilies accumulated in both late maturation stage embryos and in seedlings of B. napus. Second, a single B. napus isocitrate lyase gene, together with 3.5 kb and 1.4 kb of 5′ and 3′ flanking regions, respectively, was expressed in both embryos and seedlings of transgenic tobacco plants. The results indicated that accumulation of isocitrate lyase in late embryogeny and postgermination does not result from the alternate expression of distinct members of the gene family.     

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.     

Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.     

High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3'' untranslated region.

The high-level expression of the rat whey acidic protein (WAP) gene in transgenic mice depends on the interaction of 5'-flanking promoter sequences and intragenic sequences. Constructs containing 949 bp of promoter sequences and only 70 bp of 3'-flanking DNA were expressed at uniformly high levels, comparable to or higher than that of the endogenous gene. Although this WAP transgene was developmentally regulated, it was expressed earlier during pregnancy than was the endogenous WAP gene. Replacement of 3' sequences, including the WAP poly(A) addition site, with simian virus 40 late poly(A) sequences resulted in an approximately 20-fold reduction in the expression of WAP mRNA in the mammary gland during lactation. Nevertheless, position-independent expression of the transgene was still observed. Further deletion of 91 bp of conserved WAP 3' untranslated region (UTR) led to integration site-dependent expression. Position independence was restored following reinsertion of the WAP 3' UTR into the deleted construct at the same location, but only when the insertion was in the sense orientation. The marked differences observed between the expression levels of the 3'-end deletion constructs in transgenic mice were not seen in transfected CID 9 mammary epithelial cells. In these cells, expression of the endogenous WAP gene was dependent on the interaction of these cells with a complex extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells cultured on plastic and the precocious developmental expression of WAP in transgenic mice may reflect the absence of a negative control element(s) within these recombinant constructs.     

Secretion of biologically active human granulocyte colony-stimulating factor (G-CSF) in milk of transgenic mice

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5' and 3' flanking promoter sequences of bovine alpha-S1-casein gene. Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3' flanking region of bovine gene @CSN1S1, but differed in size of the 5' flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 microg/ml to over 1 mg/ml, in mice with construct pGCml and was low (up to 60 microg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCml and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF.     

Downstream DNA sequences are required to activate a gene expressed in the root cortex of embryos and seedlings.

We showed previously that a gene, designated AX92, which is expressed at an early stage of cortex differentiation in the root apex of oilseed rape seedlings, is also expressed in embryos. To compare AX92 gene regulation during embryo-genesis and postembryonic growth, we constructed a chimeric gene consisting of AX92 5' and 3' untranslated and flanking regions fused with a beta-glucuronidase protein coding region. We showed that the chimeric gene is active in both developing cortex cells in the root apical meristems of transgenic oilseed rape seedlings and in cortex cells at the root end of embryonic axes. To determine whether the AX92 gene is regulated by a common mechanism in embryos and seedlings, we analyzed the expression of modified chimeric genes. We showed that the AX92 chimeric gene is regulated combinatorially and that DNA sequences located 3' of the protein coding region are necessary for its activation in the root cortex of both embryos and seedlings. Our results suggest that common regulatory sequences are required to activate the gene in the embryonic and postembryonic root cortex.     

Microbody transition in greening watermelon cotyledons Double immunocytochemical labeling of isocitrate lyase and hydroxypyruvate reductase

Microbody transition during the greening of watermelon cotyledons (Citrullus vulgaris Schrad.) was studied by double immunocytochemical labeling of the glyoxysomal marker enzyme isocitrate lyase and the peroxisomal marker enzyme hydroxypyruvate reductase. In order to analyze the immunocytochemistry, developmental stages representing the glyoxysomal, microbodytransition and peroxisomal stages were chosen, taking into account the time course of enzyme activity and the amounts of the respective antigens. It was shown that during microbody transition, between 83 and 91% of all the tested microbodies contained isocitrate lyase as well as hydroxypyruvate reductase, which was significantly higher than in the glyoxysomal and peroxisomal stages of development. Comprehensive controls precluded labeling artifacts. Our results support the one-population hypothesis first proposed by Trelease et al. (1971, Plant Physiol. 48, 461–465).Abbreviations ICJ isocitrate lyase - HPR hydroxypyruvate reductase - pAg small protein A-gold complex - pAG large protein A-gold complex     

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.     

Regulation of expression of the cucumber isocitrate lyase gene in cotyledons upon seed germination and by sucrose

A 6.5 kb cucumber genomic DNA fragment containing the icl gene was introduced into Nicotiana plumbaginifolia and shown to direct isocitrate lyase (ICL) mRNA synthesis in transgenic seedlings upon germination, in a temporally regulated manner. Two putative icl promoter fragments, of 2900 and 572 bp, were subsequently linked to the GUS reporter gene and introduced into N. plumbaginifolia. Both constructs directed GUS expression after transgenic seed germination, and although the 572 bp fragment gave only 1% of the activity of the 2900 bp fragment, it directed expression in the same cotyledon-specific and temporally regulated pattern. Seedlings were transferred to darkness after 18 days growth in the light, to induce a starvation response. The 2900 bp construct was activated by starvation and repressed by exogenous sucrose, whereas the 572 bp construct was not starvation-responsive. To localize the region of the 2900 bp promoter fragment which is responsible for regulation by sucrose, further deletions were make, linked to GUS, and assayed in a cucumber protoplast transient assay system. Constructs with promoters of 2900, 2142 and 1663 bp were activated by starvation and repressed by sucrose, but promoters of 1142 and 572 bp showed no such response. We conclude that the icl gene promoter contains at least two distinct cis-acting elements, one required for the response to sucrose and the other which participates in expression upon seed germination.