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PubMed2004

Silencing of the aleurone-specific Ltp2-gus gene in transgenic rice is reversed by transgene rearrangements and loss of aberrant transcripts.

An Ltp2 promoter was isolated from barley as an aleurone-specific promoter, and its tissue specificity was maintained in transgenic rice. Expression of the Ltp2-gus gene in rice could be detected by X-gluc staining of the seeds. Previously, we reported the isolation of silenced plants in the R2 generation and the involvement of antisense gus transcripts in aleurone-specific Ltp2-gus gene silencing in transgenic rice, the L3.3 line. In the current study, we analyzed the L0.1 line, a sibling of the L3.3 line, and the partially revertant line from the L0.1 line accompanied by a transgene structural change. Strong silencing of the Ltp2-gus gene was detected over five generations in the L0.1 line. DNA and RNA analysis revealed that there were incomplete transgenes and that several aberrant RNAs that carried an antisense gus region were expressed in the L0.1 line. Determination of the transgene structure in the L0.1 line revealed that the partial antisense gus gene and the partial Ltp2 promoter region fused to the Ltp2 promoter were lacking in the revertants. RNA analysis demonstrated that the antisense gus and the promoter transcripts were produced and found in the poly(A)- fraction. Results of these analyses suggested that the observed aberrant transcripts, including antisense and promoter transcripts, were associated with Ltp2-gus silencing in the L0.1 line.

Carrier Proteins↗

PubMed1996

The expression pattern of alfalfa flavanone 3-hydroxylase promoter-gus fusion in Nicotiana benthamiana correlates with the presence of flavonoids detected in situ.

Flavanone 3-hydroxylase is an enzyme acting in the central part of the flavonoid biosynthesis pathway. It is generally encoded by a single gene and seems to have a key position for the regulation in this pathway. These two features make a single f3h promoter-gus fusion a suitable tool to study both the f3h expression and the regulation of this pathway. We present here the spatial and temporal analysis of the expression of an alfalfa flavanone 3-hydroxylase (f3h) promoter-gus fusion introduced into Nicotiana benthamiana. The Medicago sativa (alfalfa) f3h promoter directed gus expression in flowers, stems, leaves and roots. In flowers, GUS activity was observed in pollen grains, in ovules, in ovary placenta and in the epidermis, medullary parenchyma, trichomes and second cortical cellular layer surrounding the vascular bundles of the peduncle. In stems, GUS activity was detected at the same places as in the peduncle except for the medullary parenchyma. In roots, we found GUS staining in root hairs, epidermis and in the vascular bundles of the elongated zone. Finally, in leaves, the f3h promoter expressed essentially in the stalk cells of the multicellular trichomes. The expression pattern of the f3h-gus fusion was correlated to the presence of flavonoids in situ. These data indicate that this construct can be very useful to study factors controlling the production of flavonoids.

Base Sequence↗

PubMed1997

GUS expression in Arabidopsis directed by 5' regions of the pea metallothionein-like gene PsMTA.

Upstream sequences (including the first seven codons) of a metallothionein (MT)-like gene from pea, PsMTA, 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.

Arabidopsis↗

PubMed2005

Promoter trapping in Lotus japonicus reveals novel root and nodule GUS expression domains.

Agrobacterium-based transformation was used to introduce a promoter-less glucuronidase uidA gene (beta-glucuronidase; GUS) into Lotus japonicus. Transgenic plants were screened for GUS activation at different stages after inoculation with its symbiont, Mesorhizobium loti. Functional GUS fusion frequencies ranged from about 2 to 5% of the total number of transgenic lines. These lines provide excellent histological markers for tissue ontogeny analysis. Some of the activations generated GUS expression patterns that correspond to well-known tissue types, such as lateral root and nodule primordia, root tips and developing nodules (line CHEETAH). Others generated GUS activation associated with predictable but previously unknown (i) tissue types, such as the vascular bundle of the nodule (line VASCO); or (ii) expression domains, such as pericycle, nodule primordia, nodule and flower connective/vascular tissue (line FATA MORGANA) or inner root cortex cells in the vicinity of a curled root hair, nodule primordia and nodule cortex (line TIMPA). Putative members of two gene superfamilies, EH (Esp homolog) and AAA ATPase (ATPase associated with various cellular activities), were located next to the CHEETAH and VASCO insertions, respectively, and a nodulin gene, LjENOD40-2, was located next to the FATA MORGANA insertion. We utilized promoter GUS fusions to investigate the genetic regulation of LjENOD40-2 and FATA MORGANA GUS. The LjENOD40-2 promoter defined a novel expression domain and the FATA MORGANA nodule expression was reiterated by the 2 kb sequence upstream of the T-DNA insertion.

Base Sequence↗

PubMed2000

Controlled induction of GUS marked clonal sectors in Arabidopsis.

Stably transformed Arabidopsis lines in which GUS marked cell clones are readily produced in response to heat-shock have been established and characterized. Control of GUS activation is achieved by heat-shock-induced FLP recombinase activity which "switches on" expression of a GUS marker gene previously held transcriptionally silent. To obtain efficient GUS sectoring, single insert Arabidopsis lines carrying FLP recombinase under the control of a heat-shock-inducible promoter and an FLP-activatable GUS construct were generated. Analysis of GUS sectoring in lines hemizygous and homozygous for both inserts was conducted after various regimes of heat-shock were given at various developmental stages. It is shown that GUS sectoring events can be efficiently induced in most vegetative, aerial and sexual structures in Arabidopsis. Furthermore, the frequency of sectoring events, sector size and, to some extent, the tissues in which sectors are generated can be readily controlled by choice of the conditions and timing of heat-shock used.

Arabidopsis↗

PubMed2001

[1.4 kb 5' flanking region of class I patatin directs tuber-specific gus expression in potato (Solanum tuberosum L.)].

Binary vectors pPATIs (with partial signal sequence) and pPATI (without signal sequence) were constructed by fusing 1.4 kb 5' flanking regions of Class I patatin gene with GUS. Transient GUS expression was observed in in vitro tuber slices bombarded with pPATI. These constructs were then introduced into potato (cv. Desiree) via Agrobacterium tumefaciens transformation. Transgenic potato plants were confirmed by X-Gluc staining and PCR. Using in vitro tuberization system, GUS activities were assayed by fluorescence. It was shown that, in plants transformed with PATI-GUS, GUS specific activities were about 10-20 fold higher in tubers than in stems. Increased sucrose concentration could not induce PATI-GUS expression, but light enhanced PATI-GUS expression in cultured shoots.

Carboxylic Ester Hydrolases↗

PubMed2001

Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated gus transgenes in Arabidopsis.

The transgene locus KH15, which is highly susceptible to silencing in Arabidopsis thaliana, contains two inversely repeated beta-glucuronidase (gus) genes separated by a palindromic sequence and has a low GUS activity, was found to be heavily methylated in the gus coding sequence and in the center of the inverted repeat. The locus KHsb67, which is less prone to silencing, was found to be less densely methylated in the non-repetitive region that separates the inversely repeated gus genes. After the removal of one of the gus genes by Cre-mediated recombination, methylation in both loci decreased or was totally lost. Despite the presence of a 732-bp palindromic sequence in the deletion line derived from KH15, this sequence was not methylated. Whereas the KH15 locus triggers methylation of homologous gus genes when placed in trans to them, the deletion derivative did not, suggesting that the capacity for cross-talk was severely affected by disruption of the palindromic arrangement. This result suggests that the transcribed palindromic sequences are required to maintain the methylation of both symmetrically and non-symmetrically arranged cytosines.

Arabidopsis↗

PubMed2003

Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris).

Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species.

Agrostis↗

PubMed1998

Modified development in transgenic tobacco plants expressing a rolA::GUS translational fusion and subcellular localization of the fusion protein.

The rolA gene is transferred naturally by Agrobacterium rhizogenes to the genome of host plants, where it induces dramatic changes in development of transformed plants, including dwarfism and leaf wrinkling. The predicted translation product of the rolA gene is a small (11.4 kDa), basic (pI = 11.2) protein, which has no clearly significant similarity to sequences in the data bases. We have introduced into the tobacco genome a gene encoding a rolA::GUS fusion protein. Expression of this gene led to synthesis of an RNA and a protein of expected size, and the transformed plants exhibited the dwarfism and leaf wrinkling typical of rolA plants, but to a lesser degree than plants transformed with the wild-type rolA gene. The distribution of beta-glucuronidase (GUS) activity was compared in subcellular fractions of leaf extracts from plants expressing either the rolA::gus gene or a control gus construct. As expected, in the control plants, GUS activity was essentially cytosolic. In contrast, in plants expressing the rolA::gus gene the highest specific activity was associated with the plasmalemma fraction.

Amino Acid Sequence↗

PubMed1999

Ac-mediated trans-activation of the Ds element in rice (Oryza sativa L.) cells as revealed by GUS assay.

A method using particle bombardment and beta-glucuronidase (GUS) assay was applied to rice callus for detecting the excision of the maize Ds element trans-activated by the Ac transposase source. Excision of Ds biolistically introduced into rice callus resulted in the restoration of the interrupted gus gene expression, allowing visual detection of trans-activation two days after bombardment. Only the transgenic callus lines expressing the Ac transposase gene and the wild-type callus co-transformed with Ac and Ds revealed GUS activity. Frequency of excision, estimated based on the relative GUS activity, ranged from 0.3% to 2.2%. Callus lines showing different levels of Ac transcripts revealed varying excision frequencies. At the later stages of callus growth after selection for the Ac/Ds transformed lines, excision events were detected by GUS assay and confirmed by PCR and sequence analyses of the excision sites in individual colonies. GUS activity was also demonstrated in the primary regenerants from the Ac/Ds-transformed callus colonies. The method described in this study may be used as an approach for rapid detection of excision events and assessment of various factors limiting Ac/Ds activity in rice cells.

Biolistics↗

PubMed1993

The GUS gene fusion system (Escherichia coli beta-D-glucuronidase gene), a useful tool in studies of root colonization by Fusarium oxysporum.

The plant-pathogenic fungus Fusarium oxysporum was successfully transformed with the beta-D-glucuronidase gene from Escherichia coli (gusA) (GUS system) in combination with the gene for nitrate reductase (niaD) as the selectable marker. The frequency of cotransformation, as determined by GUS expression on plates containing medium supplemented with 5-bromo-4-chloro-3-indolyl glucuronide (GUS+), was very high (up to 75%). Southern hybridization analyses of GUS+ transformants revealed that single or multiple copies of the gusA gene were integrated into the genomes. High levels of GUS activity are expressed in some transformants, but activity in F. oxysporum does not appear to be correlated with the copy number of the gusA gene. Since the highest activity was found in a transformant with a single copy, it can be assumed that sequence elements of F. oxysporum integrated upstream of the gene can act as a promoter or enhancer. Expression of the gusA gene was also detected during growth of the fungus in plants, indicating that the GUS system can be used as a sensitive and easy reporter gene assay in F. oxysporum.

Cloning, Molecular↗

PubMed1993

The presequence of a precursor to the delta-subunit of sweet potato mitochondrial F1ATPase is not sufficient for the transport of beta-glucuronidase (GUS) into mitochondria of tobacco, rice and yeast cells.

A precursor to the delta-subunit of sweet potato mitochondrial F1ATPase (pre-F1 delta) has an amino-terminal (N-terminal) presequence of 45 amino acid residues and its N-terminal 18 residues may form an amphiphilic alpha-helix, which is typical of mitochondrial targeting signals [Kimura et al. (1990) J. Biol. Chem. 265: 6079]. Fusion genes consisting of sequences that encoded the 25 (DG25), 46 (DG46) and 73 (DG73) N-terminal amino acids from pre-F1 delta fused to the N-terminus of the coding sequence of bacterial beta-glucuronidase (GUS) were placed downstream of the 35S promoter of cauliflower mosaic virus and used to transform suspension-cultured tobacco cells, rice calli and tobacco plants. Fusion genes were also placed downstream of the yeast GAL10 promoter and introduced into Saccharomyces cerevisiae cells. In these transformed cells, only the DG73 GUS-fusion protein was transported into mitochondria and subjected to proteolytic cleavage of the presequence. Neither transport to mitochondria nor processing of the presequence of the DG46 GUS-fusion protein, which contained the entire presequence and the processing site, occurred in either plant or yeast cells. These results indicate that the presequence of pre-F1 delta is not sufficient for the transport of the GUS protein into mitochondria in tobacco, rice and yeast cells. The requirement for the longer polypeptide from pre-F1 delta in the transport of the GUS protein into mitochondria could be due either to the lack of sufficient information for mitochondrial targeting within the presequence or to the nature of the passenger protein, GUS, used in this study.

Amino Acid Sequence↗

PubMed1987

GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

Cloning, Molecular↗

PubMed1995

Functional analysis of a recently originating, atypical presequence: mitochondrial import and processing of GUS fusion proteins in transgenic tobacco and yeast.

A gene family of at least five members encodes the tobacco mitochondrial Rieske Fe-S protein (RISP). To determine whether all five RISPs are translocated to mitochondria, fusion proteins containing the putative presequences of tobacco RISPs and Escherichia coli beta-glucuronidase (GUS) were expressed in transgenic tobacco, and the resultant GUS proteins were localized by cell fractionation. The amino-terminal 75 and 71 residues of RISP2 and RISP3, respectively, directed GUS import into mitochondria, where fusion protein processing occurred. The amino-terminal sequence of RISP4, which contains an atypical mitochondrial presequence, can translocate the GUS protein specifically into tobacco mitochondria with apparently low efficiency. Consistent with the proposal of a conserved mechanism for protein import in plants and fungi, the tobacco RISP3 and RISP4 presequences can direct import and processing of a GUS fusion protein in yeast mitochondria. Plant presequences, however, direct mitochondrial import in yeast less efficiently than the yeast presequence, indicating subtle differences between the plant and yeast mitochondrial import machineries. Our studies show that import of RISP4 may not require positively charged amino acid residues and an amphipathic secondary structure; however, these structural properties may improve the efficiency of mitochondrial import.

Amino Acid Sequence↗

PubMed2002

An improved MUG fluorescent assay for the determination of GUS activity within transgenic tissue of woody plants.

Despite the success of the MUG fluorometric assay for quantitative analysis of beta-glucuronidase (GUS) activity within a vast array of transgenic plant species and tissues, attempts to apply this protocol for analysis of woody plants has been found to be problematic, primarily due to the interfering effects of phenolics and other secondary metabolites. Our analysis of transgenic spruce needles and poplar leaves illustrates that low tissue mass to extract volume, along with the inclusion of polyvinylpolypyrolidone and either beta-mercaptoethanol or metabisulphite, are essential for producing reliable results. The primary action of these additives was found to involve increased GUS extractability and the preservation of GUS activity during extract manipulation, but they were not completely effective in eliminating GUS enzymatic inhibitors. Normalization of GUS activity upon DNA concentration was also found to be an effective alternative to protein concentration, providing the ability to make cross-species and inter-tissue comparisons of gusA transgene activity.

Fluorescence↗

PubMed2000

Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco.

Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard procedures. This will allow for the development of routine and early screenings for the presence of small RNA species and, therefore, gene silencing in transgenic plants. We further demonstrate that the small RNA fraction can be visualized with the SYBR Green II RNA stain, isolated from a gel, labeled and used as a specific probe. Using these approaches, we have fine-mapped the sequences of the GUS gene that are represented in the small RNA fraction of a GUS-silenced tobacco line containing an inverted repeat of the GUS gene. In this tobacco line, the silencing-associated small RNA is a mixture of fragments that cover the 3' two-thirds of the GUS coding region. The 5' coding and the 3' noncoding ends of the GUS mRNA are not represented in the small RNA fraction. The RNA fragments are not likely to be a primary synthesis product of an RNA-dependent RNA polymerase, but rather degradation products from nuclease activity. Surprisingly, RNA isolated from wild-type, untransformed plants showed the presence of a similar-sized small RNA pool. This might indicate the existence of small RNA species from putative endogenous genes that are down regulated by a similar posttranscriptional gene-silencing mechanism. The possibility of isolating and labeling the small RNA pool from wild-type plants will provide a way to identify and study such putative genes.

Fluorescent Dyes↗

PubMed2002

Specific degradation of 3' regions of GUS mRNA in posttranscriptionally silenced tobacco lines may be related to 5'-3' spreading of silencing.

Target regions for posttranscriptional silencing of transgenes often reside in the 3' region of the coding sequence, although there are exceptions. To resolve if the target region is determined by the gene undergoing silencing rather than by the structure of the transgene loci or the plant genetic background, we have performed detailed analyses of target regions in three spontaneously beta-glucuronidase (GUS) silencing tobacco lines of different origin. From quantitative cosuppression experiments, we show that the main target region in all three tobacco lines is found within the 3' half of the GUS coding region but upstream of the last 200 nt. The quantities of small (21-25 nt) RNAs homologous to 5' or 3' regions of the GUS coding sequence were found to correlate approximately with the target strength of the corresponding regions. These results suggest that transgene locus structure and plant genetic background are not major determinants of silencing target regions. We also show that virus-induced gene silencing (VIGS) of GUS in Nicotiana benthamiana is induced equally effectively with Potato virus X carrying either the 5' or 3' third of the GUS coding region. This indicates that both regions can act as efficient inducers as well as targets of posttranscriptional silencing, although the 3' region is the predominant target region in the spontaneously silencing transgenic plant lines examined. Finally, we investigated spreading of the target region in the N. benthamiana plants undergoing VIGS. Surprisingly, only evidence for spreading of the target region in the 5'-3' direction was obtained. This finding may help explain why the majority of target regions examined to date lie within the 3' region of transgenes.

Gene Silencing↗

PubMed2002

NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS.

GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.

Amino Acid Sequence↗