A 470 bp WAP-promoter fragment confers lactation independent,progesterone regulated mammary-specific gene expression in transgenic mice

The ability of a 470 bp sub-fragment of the murine whey acidic protein (WAP) promoter in the context of a retroviral expression plasmid to direct gene expression to mammary epithelial cells was analysed in a number of independent transgenic mouse lines. In contrast to previous findings with the genuine 2.5 kb promoter fragment, our studies revealed a highly mammary gland-specific expression detectable only in non-lactating animals. This suggested a mainly progesterone-regulated activity of the short fragment. Therefore, transgene expression was examined in the progesterone-determined estrous cycle and during pregnancy. In accordance with in vitrodata from stably transfected cell lines, in both situations expression was upregulated at stages associated with high progesterone levels. Taken together these data provide deeper insight into WAP-promoter regulation and stress the usefulness of the shortened fragment for a lactation independent mammary-targeted expression.     

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O₂ deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8 % of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8 % of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.     

Studies on DNA—protein interactions in the upstream regulatory region of the human ε—globin gene promoter

The erythroid- and developmental stage-specific expression of the human ε-globin gene is controlled,in part,by the 5‘-flanking DNA sequence of this gene.In the present study,we have used DNA-protein binding assays to identify trans-acting factors which regulate the temporal expression of the human ε-globin gene during development.Using gel mobility shift assays and DNaseI footprinting assays,a nuclear protein factor (termed ε-SSF1) in the nuclear extracts from mouse haematopoietic tissues at d 11 and d 13 of gestation was identified.It could specifically bind to the positive control region (between-535 and -453bp) of the human ε-globin gene.We speculated that the ε-SSF1 might be an erythroid-and developmental stage-specific activator.In addition,we found another nuclear protein factor (terned ε-R1) in the nuclear extract from mouse fetal liver at d18 of gestation,which could strongly bind to the silencer region (between-392 and -177bp) of this gene.Therefore,we speculated that the ε-R1 might be an erythroid-and developmental stagespecific repressor.Our data suggest that both ε-SSF1 and ε-R1 might play important roles in developmental regulation of the human ε-globin gene expression during the early embryonic life.On the hand,we observed that the binding patterns of nuclear proteins from three cell lines (K562,HEL and Raji) to these regulatory regions were partially different.These results suggest that different trans-acting factors in K562,HEL and Raji cells might be responsible for activating or silencing the human ε-globin gene in three different cell lines.     

Transgenic expression of a Δ12-epoxygenase gene in Arabidopsis seeds inhibits accumulation of linoleic acid

The Crepis palaestina cDNA Cpal2 encodes a Δ12-epoxygenase that can catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of the napin seed-specific promoter in Arabidopsis thaliana (L.) Heynh., the seed lipids accumulated only low levels of 18:1E and also 12,13-epoxy-cis-9,15-octadec-2-enoic acid (18:2E). Despite the fact that the levels of these epoxy fatty acids comprised only up to 6.2% of the total fatty acids, there was a very marked increase in oleic acid (18:1) and decrease in linoleic (18:2) and α-linolenic (18:3) acids in these plants, indicating that endogenous Δ12-desaturation was greatly reduced in these plants. Significant between-line differences in the levels of Cpal2 mRNA were observed during seed development, but were not associated with any major variation in mRNA levels for the endogenous ArabidopsisΔ12-desaturase (Fad2). This suggests that if an unfavourable interaction occurs between the transgenic Δ12-epoxygenase and the endogenous Δ12-desaturase, which decreases the level of desaturation, it occurs at either the translational or post-translational level. We further show that the co-expression of a Δ12-desaturase gene from C. palaestina in Cpal2 transgenic Arabidopsis returns the relative proportions of the C18 seed fatty acids to normal levels and results in an almost twofold increase in total epoxy fatty acids. Received: 11 August 2000 / Accepted: 7 September 2000     

Functional analysis of OsTUB8, an anther-specific β-tubulin in rice

Microtubules play important roles in many cellular processes, such as cell division and cell elongation in plants. β-tubulins (TUB), which are one of the basic components of microtubules, are encoded by multigene family in eukaryotes and their nucleotide sequences are highly conserved in protein coding regions. OsTUB8 that was expressed in rice anthers was characterized with a multi-level approach. At the protein level, OsTUB8 was expressed mainly in anthers compared to callus, root, leaf sheath and leaf blade. In situ hybridization and GUS fusion analysis revealed that OsTUB8 was expressed in vascular bundles of anther filaments and in pollen. OsTUB8 expression was lower in the anthers of GA-deficient mutants, ‘Tanginbozu’ and ‘Akibrarewaisei’, compared to those of their respective wild types. Transgenic rice expressing OsTUB8 in an antisense orientation were suppressed in the amount of seed set upon maturity. Antisense-transgenic rice plants were 20–60% shorter compared to the vector-only control. These results suggest that OsTUB8 might be differentially expressed in rice anthers due to the action of GA, and involved in the processes of vegetative growth and seed set in rice.     

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.     

The regulatory protein MucR binds to a short DNA region located upstream of the muc R coding region in Rhizobium meliloti

The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan. The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17?kDa apparent molecular mass, corresponding to the size expected for MucR. As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene. Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site. Although binding of MucR to this site exhibited a moderate dissociation constant of $K_{\rm d} \approx 1.4 \times10^{-7}$ M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding.     

A haplotype-linked four base pair deletion upstream of the Aγ globin gene coincides with decreased gene expression

Summary During normal human development, a switch is classically observed in the relative expression of the two globin genes, the G/A ratio varying from 70/30 at birth to 40/60 by the end of the first year. An exception to this developmental pattern is linked to the presence of an XmnI restriction site at a position — 158 to the Cap site of the G gene. Another exception is observed in individuals homozygous for two easily detectable variations of the A gene: the presence of a threonine residue at codon 75 and a HindIII site within the second intron. A 4-bp deletion has been described around position — 225 in some thalassemic patients presenting with these variations. In this study, we find this deletion to be haplotypelinked in a series of 156 individuals of various ethnic origins and presenting with various normal and pathological phenotypes. In sickle cell patients heterozygous for this 4-bp deletion, the relative expression of the A genes on the two chromosomes can be measured by estimating the AT and AI chains, the former always being synthesized at a lower rate. These results suggest a functional role for the deleted sequence.     

GUS expression in Arabidopsis directed by 5′ regions of the pea metallothionein-like gene PsMT A

Upstream sequences (including the first seven codons) of a metallothionein (MT)-like gene from pea, PsMT A, were fused to GUS and introduced into Arabidopsis. High-level GUS expression was detected in the roots of plants grown on MS medium, except in regions proximal to the root apex. There was precise delineation of the root-shoot boundary. In soil-grown plants there was low GUS expression and this was absent from the more mature regions of the roots. In the aerial tissues of soil-grown plants, GUS expression was restricted to hydathodes, stipules, expanding cotyledons and the following senescent tissues: leaves, cotyledons, petals, sepals, filaments, stigmas, nectaries and siliques. A 298 bp region was shown to be required for GUS expression in roots but not for expression in vegetative aerial tissues of plants grown on MS medium. This region contains predicted ethylene-responsive elements (EREs) but similar patterns of GUS expression were detected in etr1 seedlings. GUS expression was significantly higher in roots exposed to 500 nM copper, but this increase was small in proportion to expression in roots exposed to 50 nM copper.     

Point mutations in the upstream region of the α-galactosidase A gene exon 6 in an atypical variant of Fabry disease

Summary Single point mutations in the upstream region of exon 6 of the -galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; ³⁰¹ArgGln (⁹⁰²GA) in a case that has already been published and ²⁷⁹GlnGlu (⁸³⁵CG) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; ³²⁸GlyArg (⁹⁸²GA) in the downstream region of exon 6 in one case and two combined mutations, ⁶⁶GluGln (¹⁹⁶GC)/¹¹²ArgCys (³³⁴CT), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the -galactosidase A gene.