Levels Of GUS Expression Research Articles

Analysis of the promoter of an abscisic acid responsive late embryogenesis abundant gene of Arabidopsis thaliana

Late embryogenesis abundant (lea) proteins are a diverse group of proteins present in many mono- and dicotyledonous plants. The genes encoding lea proteins are expressed in the embryo during the late stages of seed development. However, expression can also be induced in immature seeds and vegetative tissues by abscisic acid (ABA). Lea genes thus provide a model with which to study tissue-specific, developmental and hormonal regulation of expression. We used the β-glucuronidase ( iudA) reporter gene (gus) to identify functional domains in the promoter of a lea gene of Arabidopsis thaliana (AtEm1). We found that a promoter fragment extending from −182 bp to +72 bp is sufficient to direct gus expression to embryos and pollen of transgenic tobacco. Gus expression in embryos and pollen was developmentally regulated, being expressed during the late stages of seed and anther development. Comparison of different deletion constructs showed that in both tissues promoter sequences between −1443 bp and −430 bp had no effect on the level of gus expression, whilst the region between −430 bp and −182 bp is necessary for full level expression. The response to ABA was studied in seedlings of transgenic tobacco transformed with a gus gene fusion construct containing −1443 bp to +72 bp of promoter. Treatment with 50 μM ABA resulted in a 3–4-fold increase in GUS activity, indicating that ABA induction of AtEm1 acts at least in part at the level of transcription. Conserved regulatory elements were identified in the promoter of AtEm1 by sequence analysis. The possible role of these elements, and the significance of the observed gus expression in pollen are discussed.

A comparison of the activity of three cauliflower mosaic virus 35S promoters in rice seedlings and tobacco (BY-2) protoplasts by analysis of GUS reporter gene transient expression

The cauliflower mosaic virus (CaMV) 35S promoter is active in most plant species and has been used for genetic engineering to obtain strong, constitutive expression of numerous, unrelated genes. We examined the transient expression of constructs in which the uidA (GUS) reporter gene was driven by three somewhat different, independently isolated CaMV 35S promoters. These CaMV 35S promoters differed principally in 5–6 bases outside the region containing cis-acting regulatory elements, but they were identical in sequences within known regulatory elements. Plasmid DNA containing the chimeric constructs was delivered to protoplasts derived from cultured BY-2 tobacco cells by electroporation and to rice embryos by DNA uptake during imbibition. GUS enzyme activity was determined by a fluorometric method 72 h after protoplast electroporation. The rice embryos were germinated after DNA uptake during imbibition and GUS activity was determined histochemically and fluorometrically in 7-day-old seedlings. All three CaMV 35S promoters gave similar levels of GUS expression in rice seedlings, both in terms of the pattern of expression in the various seedling tissues and in terms of the total level of GUS activity per seedling. In contrast, there was nearly a 14-fold difference in the activity of the promoters in the tobacco protoplasts. Promoter activity in tobacco appeared to be strongly affected by the sequence near the transcriptional start site where the promoters differed. These results suggest that monocots and dicots may utilize somewhat different signals for transcription initiation.

Isolation of a Polyubiquitin Promoter and Its Expression in Transgenic Potato Plants

A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.

Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes

SummaryTo investigate promoter strength and the tissue‐specific patterns of expression, chimeric promoters incorporating subdomains of mannopine synthase (mas2′) and octopine synthase (ocs) promoter and activator regions were affixed to a β‐glucuronidase reporter gene and the constructions introduced into tobacco plants. Addition of a trimer of the ocs upstream activating sequence (UAS) to a mas promoter/activator region resulted in highly elevated levels of GUS activity in all tissues examined. In leaf tissue, this chimeric promoter is approximately 156‐fold and 26‐fold stronger than are the CaMV 35S and the ‘enhanced’ double CaMV 35S promoters, respectively. Expression of GUS activity directed by the mas and ocs promoters/activators is limited to specific cell types. Addition of the ocs or mas UAS to the mas or ocs promoter/activator regions modulated these expression patterns. The addition of a trimer of the ocs UAS to the mas promoter/activator region resulted in a transcriptional control element that directed GUS expression in most cell types. In addition to the strong expression in transgenic tobacco plants, this novel promoter directed higher levels of GUS expression than did the CaMV 35S promoter in transiently transformed tobacco leaf discs and suspension culture cells, as well as in cassava and cowpea explants. It is proposed that the strong promoter containing a trimer of the ocs UAS affixed to the mas promoter/activator will be useful for the very high level constitutive expression of linked genes in a wide variety of plant species.

Transient β-glucuronidase expression in Scots pine tissues derived from mature trees

Vegetative buds and bud-derived calli and suspension cells from 5- to 50-year-old Scots pines (Pinussylvestris L.) were used as targets for biolistic transformation. The gene construct used in the experiments was 35S CaMV–β-glucuronidase (GUS). The highest average level of transient GUS expression was found in suspension cells: 1229 ± 359 (mean ± SE) expressing cells per million. Transient expression was found in 35 of 44 (79%) tree genotypes studied. The expression level in buds and in calli was low: one or two spots per expressing bud. Growth-regulator pretreatment (BAP and 2,4-D) increased the number of GUS-expressing buds significantly. The high transient expression level in suspensions and the high percentage of GUS-expressing genotypes suggest that mature Scots pine tissues have potential for further transformation and genetic regulation studies.

Rice Triosephosphate Isomerase Gene 5[prime] Sequence Directs [beta]-Glucuronidase Activity in Transgenic Tobacco but Requires an Intron for Expression in Rice.

In rice (Oryza sativa L.), cytosolic triosephosphate isomerase (TPI) is encoded by a single gene. TPI catalyzes a vital step in glycolysis, and RNA blots showed that the tpi gene is expressed in all vegetative tissues (root, culm, and leaves) and in rice suspension cells. No effect of light on expression was detected, but submergence of rice seedlings resulted in elevated levels of TPI mRNA in roots and culms. The 2767-bp 5[prime] upstream sequence of the tpi gene was fused translationally with the [beta]-glucuronidase (gusA) gene, and the resulting construct, TPI-GUS, was found to express constitutive, high levels of GUS activity in transgenic tobacco (Nicotiana tabacum) plants. However, the same construct yielded no GUS activity in stably transformed rice plants, and RNA blots showed that no GUS mRNA could be detected even though stable integration of functional copies of the construct was confirmed by Southern blot and genomic polymerase chain reaction analyses. Transient assays using particle bombardment yielded high levels of GUS expression from the TPI-GUS construct in tobacco leaves, but essentially no expression in rice, barley, or maize leaves. When the first intron of the tpi gene was included in the construct (TPI-int1-GUS), transient GUS activity was routinely obtained in rice leaves, revealing that the first intron of the rice tpi gene is crucial for its expression in rice. TPI-int1-GUS also directed transient GUS expression in maize and barley leaves, but little or no activity was obtained from this construct in tobacco, tomato, or soybean leaves. These results with the rice tpi promoter are in accordance with mounting evidence that differences in gene expression exist between monocots and dicots.

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 μg per 5 × 105 protoplasts), inclusion of carrier DNA (50 μg), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3′ and 5′ to the marker gene yielded the highest levels of GUS expression.

Transient expression of the uidA gene in pollen embryoids of wheat following microprojectile bombardment

Transient GUS expression levels in microprojectile bombarded, microspore-derived embryoids of bread wheat ( Triticum aestivum cv. Pavon) were evaluated when varying the stage of embryoid development, time in culture, type of promoter, and other physical parameters. GUS expression was histochemically measured 48 h after delivery of the uidA gene. The maize ubiquitin 1 promoter provided a higher level of GUS expression than the CaMV 35S or pollen-specific Zm13 promoters. The stage of embryogenic development had little effect on transient expression up to 10 weeks in culture, after which efficiency dropped off dramatically. The source of calcium used to precipitate the DNA onto microcarriers had a profound influence and GUS expression was enhanced significantly when the calcium solution was at pH 10. Bombardment did not inhibit regeneration and GUS expression could be detected in embryoids producing roots, stems, and leaves 21 days after transformation. These results show that this system is useful for transient expression assays and suggests that the stable transformation of wheat using microspore-derived embryoids may be a practical alternative to methods involving prolonged tissue culture and regeneration from undifferentiated callus cultures.

Plastid targeting of E. coli ?-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells

Dicot and monocot chloroplast targeting peptides (CTPs) were evaluated for their effect on targeting, processing, and expression of two reporter proteins in maize cells. When tested transiently in maize leaf protoplasts, the maize ribulose bisphosphate carboxylase small subunit CTP required the inclusion of the amino terminus of mature small subunit protein to target β-glucuronidase (GUS) to the plastid. To remove this amino terminal extension from GUS after import and processing, a repeat of the native processing site was inserted between the native mature protein and the reporter protein. This repeat processing site was used with less efficiency than the native site. Parallel constructs using the Arabidopsis thaliana small subunit and maize granule-bound starch synthase CTPs also localized GUS, but varied in repeat site use and GUS expression levels. Data from the CTP fusions with GUS were generally confirmed with fusions to an allosteric variant of E. coli ADP-glucose pyrophosphorylase. Plastid targeting of this enzyme was required for starch enhancement of transgenic BMS cells.

Transgenic plants of turfgrass (Agrostis palustris Huds.) from microprojectile bombardment of embryogenic callus

Transgenic creeping bentgrass (Agrostis palustris Huds., cv. Pencross; Poaceae) plants have been obtained by microprojectile bombardment of and regeneration from embryogenic calli with a vector designed to deliver the β-glucuronidase (GUS) gene under the control of rice actin 1 5' regulatory sequences. Southern analysis of polymerase chain reaction (PCR)-amplified and restriction-digested genomic DNA of four transgenic plants regenerated from these cultures showed the unscrambled integration of the gus fragment. Northern blot analysis confirmed the expression of gus mRNA in one of the transgenic plants. Western blot analysis revealed a high level of accumulation of gus protein. Histochemical assays showed enzymatic activity of β-glucuronidase in all parts of the transgenic turfgrass plant. The order of gus expression level in different tissues of the transgenic plant is as follows: stem node > first young leaf > root tip > second / third / fourth young leaf > stem internode > root hair-zone.

Effects of tissue type and promoter strength on transient GUS expression in sugarcane following particle bombardment

Effects of tissue type and promoter strength on transient GUS expression in the sugarcane (Saccharum spp. hybrids) cultivar NCo 310 were evaluated following microprojectile bombardment of leaf explants. GUS expression was histochemically or fluorometrically measured 48 h after delivery of the uidA gene. High levels of GUS expression were obtained in leaf segments isolated from young, expanding sugarcane leaves cultured for 1, 3, or 6 d prior to bombardment. The promoter derived from the maize ubiquitin 1 gene (Ubi-1) produced significantly more GUS foci and higher GUS activity levels compared to the recombinant Emu, rice actin 1 (Act1), and CaMV 35S promoters. Our transient expression system should facilitate efforts to identify promoters and elements which will regulate desired gene expression patterns in sugarcane and aid in development of an efficient stable transformation system.

Factors influencing transient gene expression in bean (Phaseolus vulgaris L.) using an electrical particle acceleration device

The parameters influencing transient expression of the betaglucuronidase gene in bean embryonic axes, cotyledons, apical meristems and leaves were evaluated after gene delivery with an electrical particle acceleration device. A calciumspermidine procedure for coating gold particles with DNA resulted in higher levels of GUS expression with lower concentrations of gold particles compared with a calcium phosphate procedure. The DNA concentration, distance between the discharge chamber and the retaining screen and the vacuum in the apparatus also influenced gene delivery. Sections prepared for light and electron microscopy showed the localisation, within target cells, of gold particles used to deliver the DNA. Immunolocalization of foreign gene expression within cells confirmed an even distribution of gene product throughout the cell cytoplasm.

TRANSFORMATION OF GLADIOLUS FOLLOWING PARTICLE BOMBARDMENT

A genetic transformation system has been developed utilizing Gladiolus suspension cells bombarded with high-velocity DNA coated particles. Transient β-glucuronidase (GUS) expression levels from the uidA gene were measured to assess efficiency of DNA delivery following particle bombardment. We have determined that 1) non-regenerable cells express higher levels of GUS activity than regenerable cells; 2) gold particles are superior to tungsten for DNA delivery; 3) maximum GUS expression occurs in suspension cells bombarded 10-13 days after subculture; and 4) incubation of bombarded cells in osmoticum-containing medium after particle bombardment enhances GUS expression levels. Several gene promoters have been evaluated for their ability to direct GUS expression in Gladiolus and a putative stably-transformed Gladiolus callus has been isolated.

Levels and location of expression of the Agrobacterium tumefaciens pTiA6 ipt gene promoter in transgenic tobacco

The location of gene expression of the Agrobacterium tumefaciens ipt gene promoter in transgenic tobacco plants was examined using the beta-glucuronidase (GUS) reporter gene. Expression of GUS was detected in every organ and most cell types examined. The highest levels of GUS activity were found in roots. To further examine the transcriptional basis of this broad expression pattern, deletions in the 5' non-coding region of the gene were translationally fused to two promoterless reporter genes, encoding the enzymes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS). Reporter enzyme assays revealed the existence of an upstream segment required for maximal promoter function, the 5' end of which is between -442 and -408 of the Pipt ATG codon. This upstream segment is required for maximal levels of GUS expression in roots, but not in other organs, and a tobacco suspension-cultured cell line. The implications of broad ipt expression on the process of crown gall tumorigenesis are discussed.

Genetic transformation of green bean callus via Agrobacterium mediated DNA transfer

Kanamycin resistant callus was produced from leaf disc or hypocotyl expiants of green bean (Phaseolus vulgaris L.) when cultured on a defined medium containing 50 mg/l kanamycin after 4 days of co-cultivation with Agrobacterium tumefaciens strain EHA101 containing the binary vector pKYLX71GUS. The presence of neomycin phosphotransferase II (NPT-II) in crude cellular extracts from the kanamycin resistant callus was confirmed by ELISA. The expression of the ß-glucuronidase (GUS) reporter gene was confirmed by histochemical and fluorimetric analyses. Southern blot border analysis confirmed the integration of the foreign DNA. In addition to the evidence obtained from Southern analysis, the absence of Agrobacterium in the transformed callus cultures was confirmed by microscopic observation and through test cultures. Using the above protocol, bean callus cultures were also transformed with a bean chalcone synthase promoter-GUS fusion. These cultures, when treated with the elicitor glutathione, showed higher levels of GUS expression than the unelicited callus clumps.

Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts.

The transcription of zein genes in maize is tissue-specific and developmentally regulated. The 5' regulatory region of many zein genes contains two promoters, P1 and P2, lying approximately 1000 bases apart. The promoter/enhancer activity of various fragments of the two promoter regions of the zein gene E19 have been analysed by means of transient expression experiments. The results indicate that the various regions differentially affect the expression of the GUS reporter gene activity in protoplasts from tobacco leaves, maize immature endosperms and in vitro endosperm cell cultures. In tobacco protoplasts only the proximal promoter region, P2, activates GUS expression, while in endosperm culture cells only the distant promoter, P1, gives significant activity. The P1 region, both in direct and opposite orientation, stimulates a low level of GUS expression in protoplasts from immature endosperms.

A novel method for in situ screening of yeast colonies with the ?-glucuronidase reporter gene

Expression of the beta-galactosidase gene in yeast has served as a screening marker for many purposes. Here it is shown that in two yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, the beta-glucuronidase (GUS) gene can be used as an alternative marker. Since the histochemical substrate can not be taken up by yeast cells, direct colony screening of plates was found to be impossible. However, by a replica plating technique, GUS expression became visibly detectable within 10 min when the GUS gene was strongly expressed. The staining method could still be performed for expression at a 100-fold lower level, but incubation times of several hours were needed. Furthermore, specific GUS expression levels of yeast protein extracts could be quantified by a fluorometric assay which is both very simple to perform and highly sensitive. Since the GUS gene can also tolerate large N-terminal fusions, this method should be particularly attractive for studying such diverse problems as transcriptional and translational regulation or subcellular localization in yeast.

Analysis of promoter activity from an ?-zein gene 5? flanking sequence in transient expression assays

Three DNA regions required for high levels of transcription were identified by transient gene expression analysis of the 5' flanking region of a 19 kDa alpha-zein gene. For these analyses, the zein promoter region was fused to the beta-glucuronidase (GUS) gene and assayed by transient expression in carrot protoplasts. A 107-bp sequence (-114/-8) containing the TATA box resulted in low levels of GUS activity. Addition of the proximal 75 bp (-189/-114) doubled the level of GUS expression, and a further increase in expression was obtained when additional upstream sequences (-483/-226) were placed 5' of the zein promoters. Zein upstream sequences enhanced transcription independently of the -189/-114 region. Although the -189/-114 region was not essential for transcription, it was important to obtain maximum GUS activity. A 121 bp upstream sequence (-347/-226) that contains the conserved TGTAAAG sequence gave high levels of GUS activity when placed in either orientation 5' of the zein promoter sequences. However, nucleotides -347 to -309, containing the TGTAAAG sequence, could be deleted from this fragment without a significant change in GUS activity. Zein upstream sequences did not promote transcription of the GUS gene in somatic maize protoplasts. The upstream activating sequence from the cauliflower mosaic virus (CaMV) 35S promoter placed 5' of deletion mutants of the zein promoter also failed to produce GUS activity above background.

EXPRESSION OF B-GLUCURONIDASE FROM AMELANCHIER LAEVIS

Amelanchier laevis shoots were co-cultured with Agrobacterium tumefaciens carrying the binary vector pBI121, which encodes the B-glucuronidase (GUS) and neomycin phosphotransferase genes. Shoots were then rinsed briefly in liquid MS medium and plated onto culture medium containing carbenicillin. After approximately three months of culture, adventitious shoots were assayed for the presences of GUS by Southern blotting and histochemical assays. Southern analysis revealed a signal when genomic DNA from putatively transformed plants was hybridized with a probe from a segment of pBI121. Regenerated shoots also showed a deep blue color when incubated with X-gluc, although the expression was chimeric, that is, the activity appeared only in a percentage of the cells in each shoot. Callus tissue at the base of each shoot also showed high levels of GUS expression using a fluorogenic assay.

Transient gene expression in electroporated Solanum protoplasts

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts--250 V/cm; Désirée mesophyll protoplasts--225 V/cm; Désirée suspension culture protoplasts--225 V/cm; and Désirée tuber protoplasts--150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36-48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the beta-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0-10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20-30 pmol/ml) the patatin promoter directed 4-5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.