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PubMed2003

Probiotic effects of beta-glucuronidase on the peach-potato aphid Myzus persicae (Aphididae).

beta-glucuronidase (GUS) is a reporter protein commonly expressed in transgenic plants allowing the visualization of the transformed individuals. In our recent work, we showed that consumption of transformed potato plants expressing this GUS enzyme improves performance of the phloem feeding aphid Myzus persicae. Those results led us to the conclusion that the expression of GUS in potato plants might be responsible for the probiotic effect measured in feeding aphids. In the present paper, artificial diets were used to provide active GUS (10 and 500 microg ml(-1)), inactivated heated GUS (500 microg ml(-1)), glucuronic acid (10, 100 and 500 microg ml(-1)), and bovine serum albumin (500 microg ml(-1)) to M. persicae. Our results reveal that these chemicals provided as food intake might influence the biological parameters of this aphid. Experiments showed a probiotic effect of 500 microg ml(-1) GUS diet, resulting in reduced larval mortality, and increased adult reproduction period and fecundity, which led to an increased population growth potential (r(m)=0.17+/-0.01 versus r(m)=0.12+/-0.03 for aphids fed on control diet). A lower amount of added GUS led to fewer variations, biological parameters being only slightly altered (r(m)=0.14+/-0.03). Statistically similar alterations of the biological parameters were obtained when comparing aphids fed on the diet added with inactivated GUS or the non-structural bovine serum albumin protein (r(m)=0.15+/-0.02 and 0.14+/-0.03, respectively). Feeding assays conducted with glucuronic acid supplemented diets enhanced longevity and nymph production of the adult aphids and reduced larval mortality, resulting in r(m)=0.15+/-0.02 for the highest dose (500 microg ml(-1)). Although 100 microg ml(-1) glucuronate diet did not induce any effect on M. persicae (r(m)=0.12+/-0.03), aphids fed on 10 microg ml(-1) glucuronate diet exhibited unexpected reduced demographic parameters (r(m)=0.10+/-0.03). Immuno-histological analysis showed GUS labeling along the whole digestive epithelium of adults and in various tissues including embryos and bacteriocytes. These results suggest that GUS crosses through the digestive tract. Western blots performed with protein extracts of transformed potato plants expressing the gus gene showed a unique band of molecular weight 76 kDa. On the contrary, in extracts from aphids fed on transgenic potato plants or bred on GUS 500 microg ml(-1) artificial diet, several proteins of lower molecular weight were hybridized, revealing proteolysis of ingested GUS. It is concluded that GUS protein, and more precisely GUS activity, is responsible for the probiotic effects on aphid feeding. The possible pathways of induction of such physiological alterations by GUS are discussed.

Animal Feed↗

PubMed1995

Differential regulation of the expression in transgenic tobacco of the gene for beta-glucuronidase under the control of the 5'-upstream regions of two catalase genes from castor bean.

The regulatory functions of the 5'-flanking regions of two genes for catalase (cat1 and cat2) from castor bean were analyzed in transgenic tobacco plants that carried fusion constructs that included the gene for beta-glucuronidase (GUS) for Escherichia coli. Dry mature seeds from transgenic plants carrying the CAT1-GUS or CAT2-GUS constructs, in which the GUS gene was fused to the 5'-flanking region of cat1 or cat2, respectively, contained significant GUS activity, indicating that the promoters of cat1 and cat2 were active during seed development. GUS activity increased in response to germination in the seeds of transgenic tobacco that carried CAT1-GUS, as well as in those that carried CAT2-GUS. During the post-germinative stage the GUS activity directed by CAT2-GUS increased still further, whereas that directed by CAT1-GUS decreased. The changes in GUS activity in the transgenic tobacco plants that carried CAT1-GUS and CAT2-GUS were similar to the changes in the levels of transcripts of cat1 and cat2, respectively, in castor bean. The results suggest that the expression of cat1 and cat2 in the germinating seeds and post-germinative seedlings is regulated mainly at the level of transcription. However, the distribution of GUS activity among the organs of the transgenic tobacco seedlings and plantlets, which was examined by histochemical staining and by enzymatic assays of tissue extracts, was not identical to that of transcripts of cat1 and cat2 in castor bean. Histochemical analysis also revealed the interesting spatial regulation of the expression of the promoter of cat2 in the transgenic tobacco seedlings.

Base Sequence↗

PubMed1989

Two genetic elements regulate murine beta-glucuronidase synthesis following transcript accumulation.

Mutant alleles of two genetic regulatory elements, which underlie a three- to sixfold reduction in beta-glucuronidase (GUS) activity levels, distinguish mice of the H haplotype from those of the other two common GUS haplotypes, A and B. Both elements are tightly linked to the GUS structural gene over which they exert control. One (Gus-u) exerts a cis-active effect upon GUS activity levels in all tissues at all times while the other (Gus-t) regulates GUS activity in trans after the 12th postnatal day in certain tissues. While previous studies show that differences in the rate of GUS synthesis account for the combined effects of these two elements in liver of adult mice, we demonstrate the separate effects of each on GUS synthesis at times during early postnatal development when their individual expressions can be distinguished. Assessments of the relative levels of S1 nuclease protection of a radiolabeled GUS antisense RNA probe after hybridization with total liver RNA preparations from adult mice of A and H haplotypes reveal no differences. These results argue that Gus-u and Gus-t exert their control of GUS expression subsequent to the accumulation of processed GUS transcripts.

Animals↗

PubMed1996

Calcium and cGMP target distinct phytochrome-responsive elements.

Previous work using microinjection into single cells of the tomato aurea mutant demonstrated that phytochrome A-dependent activation of rbcS and chs genes was mediated by calcium and cGMP, respectively. This work sought to identify promoter cis-elements that respond to these two small molecules. Box II and Unit I, derived from rbcS-3A and chs promoters, respectively, were previously shown to function as light-responsive cis-elements. Eleven copies of Box II and four copies of Unit I were linked 5' to the -90 and -46 35 S promoters, respectively, and, both constructs were fused to the beta-glucuronidase (GUS) reporter gene. GUS activities were obtained upon coinjection of either Box II/-90GUS or Unit I/-46GUS with oat phytochrome A (phyA) and GTP gamma S, an activator of heterotrimeric G proteins. The activation of Box II/-90GUS by phyA was insensitive to the cGMP antagonist, Rp-cGMPS, although anthocyanin accumulation, but not chloroplast development, was totally blocked in the injected cells. Consistent with this result, calcium, but not cGMP, induced Box II/-90GUS activity. In contrast to Box II/-90GUS, phyA-dependent activation of Unit I/-46GUS activity was blocked by Rp-cGMPS. Moreover, cGMP, not calcium, induced Unit I/-46GUS activity. Control experiments showed that -90 GUS and -46 GUS were inactive in the presence of calcium and cGMP, respectively. These results provide evidence that Box II and Unit I are targets of the calcium and cGMP pathways, respectively. Interestingly, calcium activation of Box II/-90GUS was repressed by a high concentration of cGMP and cGMP induction of Unit I/-46GUS was blocked by a high concentration of calcium/CaM. Thus, these two small cis-elements can also serve as targets of the reciprocal control mechanisms that operate to regulate the activities of the two phyA signaling branches.

Acyltransferases↗

PubMed1995

5'-leader of a photosystem I gene in Nicotiana sylvestris, psaDb, contains a translational enhancer.

Messenger RNA primary structures responsible for translational efficiency of a photosystem I gene, psaDb, of Nicotiana sylvestris were studied using a transgenic tobacco system. The entire 5'-leader (23 base pairs) with the first four amino acid codons of the protein coding region was fused in frame with the beta-glucuronidase (GUS) gene under the control of the 35 S promoter of cauliflower mosaic virus (CaMV). This construct (CaMV::psaDb-GUS') was introduced into tobacco. GUS activity and GUS mRNA levels were determined for individual transformants, revealing that the insertion of the psaDb sequence greatly enhanced the GUS activity relative to GUS mRNA abundance. The GUS activity/GUS mRNA was 14 times higher in the CaMV::psaDb-GUS' transformants than in the control CaMV::GUS' transformants. The high GUS activity/GUS mRNA of the CaMV::psaDb-GUS' transformants was reduced 20-fold when 13 bases within the psaDb leader were altered. These 13 bases are common to the leaders of an Arabidopsis ferredoxin gene and the psaDb gene of N. sylvestris. Since GUS proteins encoded by these chimeric GUS genes have identical amino acid sequences, these results indicate that the 5'-leader of the psaDb mRNA contains a translational enhancer element.

Amino Acid Sequence↗

PubMed1988

Androgen regulation of murine beta-glucuronidase expression: identification and characterization of a nonresponse variant.

One of the major features of beta-glucuronidase (GUS) expression in inbred strains of the house mouse, Mus musculus, is the responsiveness of this enzyme to androgen stimulation in tubule cells of the kidney. Both GUS-specific and nonspecific mutations have been described which define genes that serve to control this response. During examination of the expression of GUS in the interbreeding subspecies, Mus hortulanus, a new GUS haplotype was uncovered that is characterized, in part, by a lack of GUS response to androgen stimulation in an apparently responsive kidney. Blot hybridization analyses of kidney RNA with a radiolabeled murine GUS cDNA shows this lack of response to be reflected in GUS mRNA levels. The difference in heat stability of GUS activity between M. hortulanus and a responsive inbred strain, ICR/Ha, was utilized to assess the contribution of each parent to kidney levels of GUS in androgen-treated and -untreated F1 progeny of these strains. The results, together with preliminary genetic studies, suggest that the element controlling this responsiveness (or the lack thereof) is cis-active and tightly linked to the GUS structural gene on chromosome 5. It is not known whether this element is identical to another GUS-specific, cis-active element, Gus-r, which also controls the androgen response of GUS in mouse kidney.

Androgens↗

PubMed2004

Developmental and cell-specific expression of ZWICHEL is regulated by the intron and exon sequences of its gene.

Functional studies with ZWICHEL ( ZWI ), which encodes a Ca(2+)-calmodulin-regulated kinesin, have shown its involvement in trichome morphogenesis and cell division. To identify regulatory regions that control the ZWI expression pattern, we generated transgenic Arabidopsis plants with a GUS reporter driven by different lengths of the ZWI gene 5' region alone or 5' and 3' regions together. The 5' fusions contain varying lengths of the coding and non-coding regions of beta - HYDROXYISOBUTYRYL-CoA HYDROLASE 1 ( CHY1 ), which is upstream of ZWI, and a 162 bp intergenic region. In transgenic plants with 5' 460::GUS, GUS activity was observed primarily in the root hairs whereas transgenic plants with an additional 5' 266 bp region from the CHY1 gene (5' 726::GUS) showed strong GUS accumulation in the entire root including root hairs and root tip, calli and at various developmental stages in trichomes and pollen. However, very little GUS accumulation was detected in roots of dark-grown or root tips of cold-treated seedlings with 5' ZWI constructs. These results were further confirmed by quantifying GUS enzyme activity and transcripts in these seedlings. Calli and pollen transformed with the 5' distal 268 bp fused in antisense orientation to the proximal 460 bp did not show GUS expression. Further, IAA-treated dark-grown seedlings with 726::GUS, but not with 460::GUS, showed high GUS expression in specific regions (outer layer 2a cells) at the base of the lateral roots. The ZWI 3' region (3 kb) did not influence the GUS expression pattern driven by the 5' 726 bp. The absence of CHY1 transcripts in the chy1-2 mutant did not alter either ZWI expression or ZWI-mediated trichome morphogenesis. Thus, our data suggest that the 3' part of the CHY1 gene contains regulatory elements that control ZWI gene expression in dividing cells and other cells that exhibit polarized growth such as root hairs, pollen and trichomes. This is the first evidence that the regulatory regions conferring developmental and cell-specific expression of a gene reside in the introns and exons of its upstream protein-coding gene.

5' Flanking Region↗

PubMed1997

Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.

Animals↗

PubMed2006

Roles of salicylic acid-responsive cis-acting elements and W-boxes in salicylic acid induction of VCH3 promoter in transgenic tobaccos.

A salicylic acid (SA)-inducible VCH3 promoter was recently identified from grapevine (Vitis amurensis) that contains two inverse SA-responsive cis-acting elements and four W-boxes. To further demonstrate the roles of these elements, four fragments with lengths from -1187, -892, -589, -276 to +7 bp were fused with the b-glucuronidase (GUS) reporter gene and transferred to Nicotiana tobacum, together with another four VCH3 promoter fragments with mutation in the two inverse SA-responsive elements. The functions of each promoter fragment were examined by analysis of GUS activity in the transgenic tobacco root treated with SA. Enhanced GUS activity was shown in the roots of transgenic tobaccos with the VCH3 (-1187)-GUS construct containing two SA-responsive cis-acting elements and four W-boxes. However, GUS activity directed by the VCH3 (-892)-GUS construct, containing one SA cis-acting element and four W-boxes, was reduced by up to 35% compared with that in tobaccos transformed with the VCH3 (-1187)-GUS construct, indicating that the SA cis-acting element plays an important role in SA induction of the VCH3 promoter. Neither the m2VCH3 (-1187)-GUS nor the mVCH3 (-892)-GUS construct, with mutation on the SA-responsive elements, abolished the expression of GUS activity, demonstrating that the W-boxes in the VCH3 promoter are also involved in SA induction. Histochemical analysis of GUS activity directed by each of the eight VCH3 promoter fragments showed that GUS was expressed specifically in vascular tissue. It was concluded that both the SA-responsive cis-acting elements and the W-boxes are important for the SA induction of the VCH3 promoter. This promoter might have a potential use in plant genetic engineering.

Gene Deletion↗

PubMed1989

Antisense RNA inhibition of beta-glucuronidase gene expression in transgenic tobacco plants.

Antisense RNA was used to specifically inhibit the expression of a GUS gene introduced in a transgenic plant. A tobacco transformant containing a single intact copy of the GUS gene and showing relatively high constitutive levels of GUS activity (GUS +) was re-transformed with an Agrobacterium Ti-derived binary vector containing an antisense version of this reporter gene. The sense and antisense GUS genes were each under the regulation of the CaMV 35S promoter. Re-transformed plants contained 1-5 copies of the antisense construct and all showed a greater than 90% reduction in GUS activity relative to the original GUS + plant. This reduction in GUS activity correlated closely with the levels of GUS enzyme and steady state GUS mRNA observed in these plants. The relatively low levels of sense and antisense GUS transcripts found in the re-transformed plants may indicate a rapid degradation of the RNA:RNA duplex in the cell.

Gene Expression↗

PubMed1990

Promoter sequences from a maize pollen-specific gene direct tissue-specific transcription in tobacco.

A set of 5' promoter deletions from Zmg13, a genomic clone of a pollen-specific gene of maize, has been transcriptionally fused to a beta-glucuronidase (GUS) reporter gene in the binary vector pBI101. Tobacco leaf disks were transformed and mature plants analyzed for GUS activity directed by the Zmg13 promoter constructs. Transgenic plants containing the 375 bp Zmg13 sequence from -314 to +61 relative to the transcription start site transcribed GUS RNA and expressed active GUS enzyme in mature pollen but not in leaves. Plants transformed with a 35S CaMV promoter-GUS transcriptional fusion expressed GUS RNA in leaves but not in pollen. Neither GUS RNA or active enzyme could be detected in pollen or leaves from plants containing a 124 bp Zmg13-GUS transcriptional fusion missing the putative Zmg13 TATA box. No GUS RNA or enzyme expression was not detected in non-transformed tobacco. RNA and GUS histochemical analysis of the T1 generation confirmed that the temporal expression pattern of Zmg13-GUS transcription in tobacco followed that of the native gene in maize and that the Zmg13 promoter sequences from the maize gene are able correctly to direct genetically stable, tissue-specific gene expression in transgenic tobacco plants.

Base Sequence↗

PubMed1997

Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato.

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the beta-glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.

Abscisic Acid↗

PubMed1989

DNA determinants of structural and regulatory variation within the murine beta-glucuronidase gene complex.

The murine beta-glucuronidase (GUS) gene complex, [Gus], encompasses the GUS structural element, Gus-s, and a set of regulatory elements which serve to modulate Gus-s expression. Three common GUS haplotypes representing virtually all inbred strains of laboratory mice have been compared with respect to GUS mRNA sequence. Results of such comparisons revealed sequence variations which target the location of one of the GUS regulatory elements to sequences within Gus-s and which account for known electrophoretic and heat stability differences among GUS allozymes of the three common GUS haplotypes.

Alleles↗

PubMed1999

A clinical and serological comparison of group A versus non-group A streptococcal reactive arthritis and throat culture negative cases of post-streptococcal reactive arthritis.

OBJECTIVE: To identify clinical and serological differences of patients with reactive arthritis after infection with Lancefield group A beta-haemolytic streptococci (GAS), compared with non-group A-that is, group C or G streptococci (NGAS:GCS/GGS), and a group of culture negative or unidentified streptococci (GUS). METHODS: A prospective study of consecutive patients with reactive arthritis after serologically or culture confirmed infection with beta-haemolytic streptococci, presenting to the outpatient department of rheumatology from January 1992 until January 1998. Alternative causes for reactive arthritis were excluded. Main outcome measures were clinical and serological characteristics including antistreptolysine-O (ASO) and antideoxyribonuclease-B (antiDNase-B) antibody titres. RESULTS: 41 patients (female/male ratio 22/19; mean (SD) age 38 (13) years) with reactive arthritis were included. Culture of throat swab was positive in 13 cases (32%): 6 (15%) GAS, 7 NGAS (17%), that is, 5 (12%) GCS, 2 (5%) GGS. In 28 cases throat culture remained negative resulting in a group of unidentified streptococci; antibiotic pre-treatment had been given by the general practitioner in 18 cases (64%). Arthritis was non-migratory, the number of arthritic joints in GAS and NGAS was similar, whereas in NGAS patients fewer joints were involved than in GUS: mean (SEM) 36 swollen joint index: 3.3 (1.0) in NGAS v 5.6 (1.0) in GUS (p<0.005); 28 swollen joint index: 2.9 (1.0) in NGAS v 4.3 (0.8) in GUS (p<0.05). Extra-articular manifestations-that is, erythema nodosum/ multiforme, AV conduction block or hepatitis-were observed after GAS or GUS infection, but not after NGAS infection. ASO and/or antiDNase-B rose significantly in all patients. The maximal titres for ASO and antiDNase-B in 41 PSRA patients were: mean (SEM) 1242 (232) U/l and 890 (100) U/l respectively; the maximal ASO titres were similar in the three groups: mean (SEM) 1125 (185) in GAS, 625 (160) in NGAS (GAS v NGAS: p=0.17), and 1430 (320) U/l in GUS (NGAS v GUS: p=0.10). AntiDNase-B titres were: mean (SEM) 1075 (180) in GAS, 375 (105) in NGAS (GAS v NGAS: p<0.01), and 995 (125) U/l in GUS (NGAS v GUS: p<0.005). ASO: antiDNase-B ratios were: mean (SEM) 0.89 (0.21) in GAS, 2.60 (0.76) in NGAS (GAS v NGAS: p<0.05), and 1.43 (0.28) in GUS (NGAS v GUS: p=0.12). CONCLUSION: Post-streptococcal reactive arthritis occurs not infrequently. Differentiation of PSRA based on the causative streptococcal strain is frequently thwarted by negative throat cultures. Sometimes extra-articular manifestations are present that exclude NGAS as the causative organism. Serologically, lower antiDNase-B titres may be indicative for primary NGAS infection; the ASO/antiDNase-B ratio may be of additive value for differentiation in cases of a negative throat culture: the higher ASO/antiDNase-B ratios suggesting primary NGAS infection. In reactive arthritis, serological monitoring consisting of a simultaneous titration of antiDNase-B and ASO, seems to be of clinical importance to trace GAS induced cases, especially when throat cultures remain negative.

Adult↗

PubMed2005

High-level transient expression of recombinant protein in lettuce.

Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations < or = 100 ppm in the vacuum infiltration did not affect GUS expression, while infiltration with an A. tumefaciens density of 10(7) and 10(8) colony forming units/mL, incubation at 29 degrees C, and a surfactant concentration of 1,000 ppm significantly reduced expression. Incubation in continuous light caused lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta.

Agrobacterium tumefaciens↗

PubMed1991

Construction of expression vectors based on the rice actin 1 (Act1) 5' region for use in monocot transformation.

It has been previously reported that the 5' region of the rice actin 1 gene (Act1) promoted high-level expression of a beta-glucuronidase reporter gene (Gus) in transformed rice cells. In this paper we describe the construction of Act1-based expression vectors for use in monocot transformation. As part of the development of these vectors, we have evaluated the influence of the Act1 first intron, the Act1-Gus junction-encoded N-terminal amino acids, and the sequence context surrounding the Act1 and Gus translation initiation site on Act1-Gus gene expression in rice and maize cells. We have found that addition of Act1 intron 1 to the transcription unit of a Gus reporter gene under control of the cauliflower mosaic virus (CaMV) 35S promoter stimulated GUS activity more than 10-fold in transformed rice cells. Optimization of the sequence context around the Gus translation initiation site resulted in a 4-fold stimulation of Gus expression in transformed rice cells. By utilizing both the Act1 intron 1 and optimized Gus translation initiation site, a 40-fold stimulation in Gus expression from the CaMV 35S promoter has been achieved in transformed rice cells; very similar results were obtained in transformed maize cells. Taken together these results suggest that the Act1-based expression vectors described here should promote the expression of foreign genes in most, if not all, transformed monocot cells to levels that have not previously been attainable with alternative expression vectors.

Actins↗

PubMed1999

Growth hormone and insulin-like growth factor-I enhance beta-glucuronidase gene activation by androgen in mouse kidney.

Beta-glucuronidase (GUS) is a lysosomal enzyme that, in mouse kidney, is subject to control by multiple hormones: androgen, which increases GUS transcription; estrogen, which antagonizes androgen-mediated stimulation of GUS; and growth hormone (GH), which appears to be necessary for the full androgen effect. Neither estrogen nor GH affects GUS in the absence of androgen. In hypophysectomized or pituitary dwarf mice the reduced androgen stimulation of GUS can be partially restored with GH treatment. Androgen-induced GUS mRNA increased significantly with intermittent GH, compared to no GH or continuous GH. Intact mice subjected to continuous infusion of GH showed a depressed androgen effect on GUS similar to that seen in GH-deficient mice. Thus, pulsatile GH is required for the full androgen response. Insulin-like growth factor-I (IGF-I) also restored GUS induction by androgen in GH-deficient mice. We conclude that GH enhances the effect of androgen on the GUS gene via IGF-I. Using transgenic mice, we have also identified a genetic variant of the GUS gene that is insensitive to GH enhancement of the androgen effect.

Animals↗

PubMed1996

Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation.

An electroporation-mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203-GUS, which consists of coding regions for beta-glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203-GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203-GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti-GUS antibody confirmed the existence of GUS protein in chloroplasts electroporated with chloroplast-specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS protein in treated chloroplasts was further confirmed by Western blot analysis.

Cell Fractionation↗