Chemistry: molecular biology and microbiology – Vector – per se
Patent
1994-01-21
1997-06-17
Chereskin, Che S.
Chemistry: molecular biology and microbiology
Vector, per se
435 697, 536 232, 536 234, 536 243, C12N 1562
Patent
active
056396635
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Transcriptional and translational fusions to reporter genes whose products can be easily assayed offer powerful approaches to studying gene structure, expression, regulation, gene product assembly, transport and compartmentalization (Rosenberg et al., Science 222:734-739, 1983; Bonnerot et al., Proc. Nat. Acad. Sci. U.S.A. 84:6795-6799, 1987; Finnegan et al., The Plant Cell 1:757-764, 1989). Some of these gene fusions have been applied in more wide-ranging studies such as discerning cell lineage during development and purifying gene products (Germino et al., Proc. Nat. Acad. Sci. U.S.A. 80:6848-6852, 1983; Silhavy and Beckwith, Microbiol Rev. 49:398-418, 1985; Scholtissek and Grosse, Gene 62:55-64, 1988). Pioneered with E. coli .beta.-galactosidase (lacZ), gene fusions have been adapted to other organisms including yeast, animals and plants. While transcriptional fusions might be construed as straightforward, translational fusions require reporters which can function despite covalent addition of extraneous polypeptides to their amino- or carboxy-terminus.
Some genetic markers impart a selectable phenotype such as antibiotic resistance while other markers specify an enzymatic reporter activity for which there is a colorimetric or luminescence assay, making them suitable in combination for selection and screening. Single genetic markers which provide both of these features would be particularly useful.
The following references constitute background art: symposia on molecular and cellular biology p. 279, abstr R115.
SUMMARY OF THE INVENTION
This invention pertains to genetic markers which have a biochemically assayable (reporter) activity and confer a conditionally selectable growth advantage. The genetic markers are fused genes comprising a first structural gene which encodes the biochemically assayable product and a second, different structural gene which encodes a product whose activity confers the selectable growth advantage in a transformed cell. The translational product of the fused gene is a single contiguous polypeptide (fusion protein) which exhibits the activities of both gene products. In a preferred embodiment, the first structural gene encodes an enzyme that acts on a chromogenic substrate, such as the enzyme .beta.-glucuronidase (GUS), and the second structural gene encodes an enzyme which confers antibiotic resistance to a transformed cell, such as the enzyme neomycin phosphotransferase (NPT-II) which confers resistance to several aminoglycoside antibiotics.
The genetic markers of this invention can be adapted for use in prokaryotic and eukaryotic cells, including yeast, animal and plant cells. They provide for powerful positive genetic selection and facile, sensitive biochemical and histochemical detection in transformed cells. They facilitate genetic selection of transformed cells and permit subsequent spatial localization and quantitative estimation of gene activity. In addition, the markers can be used to probe for and recover novel genes and genetic regulatory elements such as promoters.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of a fusion between the carboxy-terminus of GUS and the amino-terminus of NPT-II.
FIGS. 2A and B show the comigration of GUS and NPT-II activities in extracts of DH5.alpha. cells transformed with a plasmid carrying the gus::npt-II fused gene on molecular sieve chromatography.
FIGS. 3A and B show immunoblot analysis of E. coil-produced GUS and GUS::NPT-II fusion proteins.
FIGS. 4A and B show the elution profile of GUS and NPT-II activity in extracts of tobacco plants transformed with a plasmid carrying the gus::npt-II fusion gene on gel permeation chromatography.
FIGS. 5A and B show the immunodetection of GUS and NPT-II polypeptides in leaf extracts from the transgenic tobacco plants.
FIG. 6 shows gel electrophoretic analysis of GUS activity in tobacco plants.
FIG. 7 is a schematic diagram of a probe designed for insertional tagging of plant promoters through T-DNA mediated transformation.
FIG. 8 is a
REFERENCES:
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Cohen in The Enzymes (Boyer) Academic Press, NY, 1970, pp. 147-150 178-185, and 194-197.
Richardson (1981) Advances in Protein Chemistry vol. 34: pp. 253-339.
Jobling et al. (1987) Nature vol. 325: pp. 623-624.
Vaeck et al (1987) Nature vol. 328, pp. 33-37.
Teeri et al (1986) The EMBO Journal vol. 5(8): pp. 1755-1760.
Jefferson et al. (1987) The EMBO Journal vol. 6(13): pp. 3901-3907.
Silhavy et al (1985) Microbiological Reviews vol. 49(4): pp. 398-418.
Crosby William L.
Datla Raju S.
Hammerlindl Joe K.
Selvaraj Gopalan
Anderson J. Wayne
Chereskin Che S.
National Research Council of Canada
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