Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

Alwen, Anna; Moreno, Rosa María Benito; Vicente, Óscar; Heberle‐Bors, Erwin

doi:10.1007/bf02513023

Cited by 39 publications

(27 citation statements)

References 21 publications

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“…The results indicate that GusA is a very stable enzyme that is more active at acidic pHs and higher temperatures. The Lactobacillus enzyme is considerably more active at acidic pHs than what has been reported for the E. coli enzyme (5,28). This difference is not entirely surprising considering that the lactic acid bacteria generally inhabit a more acidic environment and maintain a lower intracellular pH than the enterobacteria.…”

Section: Discussionmentioning

confidence: 79%

Identification and Cloning of gusA , Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH

Russell¹,

Klaenhammer

2001

Appl Environ Microbiol

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The gusA gene, encoding a new ␤-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a ␤-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored ␤-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to ␤-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a ␤-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified ␤-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested.

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Section: Discussionmentioning

confidence: 79%

Identification and Cloning of gusA , Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH

Russell¹,

Klaenhammer

2001

Appl Environ Microbiol

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“…Cambial discs were incubated in a water bath in GUS solution at 55°C for 10 minutes prior to being placed upright in the dark on a rotary shaker (150 rpm) at 37°C overnight. The GUS solution was then replaced with 70% ethanol and samples were stored at 4°C until assessment (Spokevicius et al 2005, Alwen et al 1992.…”

Section: Plant Materialsmentioning

confidence: 99%

Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues

Creux

Bossinger

Myburg

et al. 2012

Planta

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The increasing focus on plantation forestry as a renewable source of cellulosic biomass has emphasized the need for tools to study the unique biology of woody genera such as Eucalyptus, Populus and Pinus. The domestication of these woody crops is hampered by long generation times, and breeders are now looking to molecular approaches such as markerassisted breeding and genetic modification to accelerate tree improvement. Much of what is known about genes involved in the growth and development of plants has come from studies of herbaceous models such as Arabidopsis and rice. However, transferring this information to woody plants often proves difficult, especially for genes expressed in woody stems. Here we report the use of Induced Somatic Sector Analysis (ISSA) for characterization of promoter expression patterns directly in the stems of Populus and Eucalyptus trees. As a case study, we used previously characterized primary and secondary cell wall related cellulose synthase (CesA) promoters cloned from Eucalyptus grandis. We show that ISSA can be used to elucidate the phloem and xylem expression patterns of the CesA genes in Eucalyptus and Populus stems, but also show that the staining patterns differ in Eucalyptus and Populus stems. These findings show that ISSA is an efficient approach to investigate promoter function in the developmental context of woody plant tissues and raise questions about the suitability of heterologous promoters for genetic manipulation in plant species.

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“…Under the conditions used, no endogenous GUS activity was detected in either assay (ref. 24 and unpublished data). Histochemical assays with in vivo pollen were performed after isolating them from the surrounding anther tissues (25).…”

Section: Methodsmentioning

confidence: 68%

Pollen selection: a transgenic reconstruction approach.

Touraev

Fink²,

Stöger³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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A transgenic reconstruction experiment has been performed to determine the feasibility of male gametophytic selection to enhance transmission of genes to the next sporophytic generation. For tobacco pollen from a transgenic plant containing a single hygromycin-resistance (hygromycin phosphotransferase, hpt-) gene under control of the dc3 promoter, which is active in both sporophytic and gametophytic tissues, 3 days of in vitro maturation in hygromycincontaining medium was sufficient to result in a 50% reduction of germinating pollen, as expected for meiotic segregation of a single locus insert. Pollination of wild-type plants with the selected pollen yielded 100%o transgenic offspring, as determined by the activity of the linked kanamycin-resistance gene-present within the same transferred T-DNA bordersunder control of the nos promoter. This is direct proof that selection acting on male gametophytes can be a means to alter the frequency of genes in the progeny.The plant life cycle consists of two distinct phases, the diploid sporophytic"and the haploid gametophytic phase. In angiosperms, the haploid phase is reduced, and the male and female gametophytes are enclosed in the respective sex organs of the sporophyte. Male and female gametophytes (i.e., pollen and embryo sacs) are formed from haploid spores, the direct products of meiosis, and the sporophytic phase starts again after the fusion of the two pollen-contained sperms with the egg and polar cells of the embryo sac in a double fertilization event. In spite of the large morphological and physiological differences, a large overlap of gene expression has been demonstrated between the sporophytic and gametophytic phase on the protein and mRNA level (for reviews, see refs. 1 and 2).These data provided the theoretical basis for the early pollen selection experiments (for review, see ref.2). The idea was that if such an overlap exists, it should be possible to exert selection pressures on pollen to increase the frequency of responsive genes in the subsequent sporophytic generation. Angiosperm pollen has some features that makes pollen selection a very attractive method of plant breeding: large population size, haploid nature of the cells, and independence from the maternal plant (3).A variety of selection pressures such as drugs, heat, water, cold temperature, and other stresses have been applied to mature pollen just before pollination, and nonstressed plants have been pollinated with the selected pollen in anticipation of obtaining progeny in which an increased frequency of resistant individuals can be found (for review, see ref. 4). Despite a few positive reports (5-9), the feasibility of pollen selection is still under question. This is mainly due to the problem of discriminating between gametophytic and sporophytic effects on pollen development (resulting from pollen-and antherexpressed genes, respectively; refs. 2 and 9) and a lack of appropriate selection schemes. The developmental windowThe publication costs of this article were defrayed in part by...

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Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

Cited by 39 publications

References 21 publications

Identification and Cloning of gusA , Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH

Identification and Cloning of gusA , Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH

Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues

Pollen selection: a transgenic reconstruction approach.

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