Glucuronidase Assay Research Articles

Agrobacterium-mediated transformation of white mustard (Sinapis alba L.) and regeneration of transgenic plants

A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7-9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and β-glucuronidase. Shoots are regenerated from callus-forming explants within 3-4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14-16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical β glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.

Expression of the [beta]-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.

A [beta]-glucuronidase (GUS) gene that is under the control of the anther-specific LAT52 promoter of tomato (Lycopersicon esculentum) and the nopaline synthetase polyadenylation terminator was successfully expressed in pollen of Lilium longiflorum, Nicotiana tabacum, Nicotiana rustica, and Paeonia lactiflora using a pneumatic particle gun. The GUS gene in plasmid pBI221 was also expressed, to a lesser extent, in pollen of all of these species. The presence of methanol in the substrate solution for histochemical GUS assay and the incubation time in this solution influenced successful detection of GUS expression in bombarded pollen. Cytological analysis of GUS-expressing pollen of lily showed that introduced gold particles were seen in intracellular compartments of pollen, including the vegetative cytoplasm, vegetative nucleus, and generative cytoplasm.

Cauliflower Mosaic Virus Gene VI Controls Translation from Dicistronic Expression Units in Transgenic Arabidopsis Plants.

Transformed Arabidopsis plants were used to study the effect of the cauliflower mosaic virus (CaMV) inclusion body protein on translation of dicistronic RNA. Reporter plants contain a dicistronic transcription unit with CaMV open reading frame VII (ORF VII) as the first and the [beta]-glucuronidase (GUS) reporter ORF as the second cistron. "Transactivator plants" contain CaMV ORF VI under the control of the strong CaMV 35S promoter. The transactivator plants were difficult to regenerate and showed an abnormal phenotype. Expression of GUS activity in the reporter plants was very low, but high GUS activity could be induced by introduction of gene VI, either by crossing with plants containing gene VI as a transgene or by infection with CaMV. Histological GUS assays showed that transactivation occurred in all types of tissue and at all developmental stages. The practical implications of the induction of GUS expression from the dicistronic unit by virus infection are discussed.

30] Nondestructive assay for β-glucuronidase in culture media of plant tissue cultures

The development of the β- glucuronidase (GUS) gene fusion system and the proliferation of many GUS gene (uidA) constructions have made the screening for transformed plant tissues easy and rapid. Although milligram amounts of tissue are required, the method is destructive and there are times when valuable tissue must be sacrificed for the assay. This destructive aspect of the assay can be a drawback for some applications of the GUS reporter system. The nondestructive method is a simple derivation of the standard GUS fluorometric assay, using methylumbelliferyl glucuronide (MUG) described in this chapter. In this derivation, the substrate is placed in the culture medium for the assay rather than in a tissue extract. This procedure was developed to avoid extraction of tissues in screening progeny. The MUG can be used in the phosphate buffer described for the GUS colorimetric assay, as well as in the standard GUS lysis buffer. This variation of the GUS assay is easily performed on the spent culture medium after routine reculture of the tissue.

Intron Splicing and Intron-mediated Enhanced Expression in Monocots

Publisher Summary The basic recognition sites (5 ‘ , 3‘, and branchpoint) for intron splicing in plants do not differ significantly from those of vertebrates. It is also demonstrated, by the mutations of splice-site recognition sequences, that the 5 ‘ GT or 3‘ AG dinucleotides are essential for proper splicing in monocots. The differences in base composition observed in monocot and dicot introns are described in this chapter. These differences could explain some of the reports of inefficient splicing or nonsplicing observed in dicot cells with genes containing monocot introns. The reports of splicing difficulties between vertebrate and plant introns in heterologous systems may be attributed to the lack of a good polypyrimidine sequence adjacent to the 3‘ splice-site in the plant introns. Moreover, previous reports by other investigators have broadly generalized the observations of inefficient or incorrect splicing in dicot cells (with vertebrate introns) to be indicative of results in all plants. The chapter also presents experimental data indicating that (1) splicing is required to achieve enhanced expression of reporter genes in protoplasts and (2) the various introns are spliced with differing efficiencies. These data also demonstrates that the degree of enhanced β- glucuronidase (GUS) expression obtained in assays is directly correlated with the extent of RNA splicing. Utilizing the transient GUS assay system as a measure of splicing efficiency, it has been found that maize cells are very proficient at processing introns from both monocot and dicot sources, whereas tobacco or carrot cells are not.

Glucuronidase Assay in a Rapid MPN Determination for Recovery of Escherichia coli from Selected Foods

Glucuronidase is present in most strains of Escherichia coli but absent in most other enteric microorganisms; therefore, an assay for this enzyme is useful for determining the presence of the organism. The substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) is incorporated into either lauryl tryptose (LT) broth or EC medium; the inoculated tubes are then incubated under specified conditions and examined under longwave UV light for the presence of a fluorogenic glucuronidase end product. When compared with the 10-day most probable number (MPN) procedure of AOAC, the LT-MUG and the EC-MUG tests required 24 and 96 h, respectively, and gave comparable mean log MPN values for samples of crabmeat, sunflower kernels, and walnut pieces. However, false-positive and false-negative reactions were observed with foods tested by both of these rapid methods. Overall, method sensitivity was not compromised by using the LT-MUG rather than the EC-MUG method. Incorporation of 25 micrograms MUG/mL into LT broth resulted in diminished fluorescence of positive reactions, whereas MUG concentrations of 50 and 100 micrograms/mL provided decisive fluorogenic reactions.