Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-08
2002-05-21
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023200, C536S024100, C435S069100, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06391547
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to forms of microbial &bgr;-glucuronidase that are directed to specific cell compartments, and more specifically to a secreted form of &bgr;-glucuronidase and uses of these &bgr;-glucuronidases thereof.
BACKGROUND OF THE INVENTION
The natural habitat of
E. coli
is the gut, and the &bgr;-glucuronidase (GUS) activity of
E. coli
plays a specific and very important role in its natural history. The gut is a rich source of glucuronic acid compounds, providing a carbon source that can be efficiently exploited by
E. coli
. Glucuronide substrates are taken up by
E. coli
via a specific transporter, the glucuronide permease (U.S. Pat. Nos. 5,288,463 and 5,432,081), and cleaved by &bgr;-glucuronidase. The glucuronic acid residue thus released is used as a carbon source. In general, the aglycon component of the glucuronide substrate is not used by
E. coli
and passes back across the bacterial membrane into the gut to be reabsorbed into the bloodstream and undergo glucuronidation in the liver, which begins the cycle again.
In
E. coli
, &bgr;-glucuronidase is encoded by the gusA gene (Novel and Novel
Mol. Gen. Genet
. 120:319-335, 1973), which is one member of an operon comprising three protein-encoding genes. The second gene, gusB, encodes a specific permease (PER) for &bgr;-glucuronides. The third gene, gusC, encodes an outer membrane protein (MOP) of approximately 50 kDa that facilitates access of glucuronides to the permease located in the inner membrane. The principle repressor for the GUS operon, gusR, maps immediately upstream of the operon.
&bgr;-glucuronidase activity is expressed in almost all tissues of all vertebrates and many mollusks (Levvy and Conchie, 1966). In addition, the free-living soil nematode,
Caenorhabditis elegans
, has an endogenous &bgr;-glucuronidase activity (Sebastiani et al, 1987; Jefferson et al, 1987), which occurs at low levels in the intestine of the worm. The enzyme has been purified from many mammalian sources (e.g. Tomino et al, 1975) and forms a homotetrameric structure with a subunit molecular weight of approximately 70 kDa.
The vertebrate enzyme is synthesized with a signal sequence at the amino terminus, then transported to and glycosylated within the endoplasmic reticulum, and ultimately localized intracellularly within vacuoles. If any of the mammalian enzyme is secreted, it probably contributes little to the total activity as the enzyme is relatively unstable. Thus, for use in medical diagnostics (e.g., drug testing) and transgenic constructions, the
E. coli
enzyme is preferred because it is much more active and stable than the mammalian enzyme against most biosynthetically derived &bgr;-glucuronides (Tomasic and Keglevic, 1973; Levvy and Conchie, 1966).
Production of GUS for use in in vitro assays, such as medical diagnostics, is costly and requires extensive manipulation as GUS must be recovered from cell lysates. A secreted form of GUS would reduce manufacturing expenses, however, attempts to cause secretion have been unsuccessful. In addition, for use in transgenics, the current GUS system has somewhat limited utility because enzymatic activity is detected intracellularly by deposition of toxic calorimetric products during the staining or detection of GUS. Moreover, in cells that do not express a glucuronide permease, the cells must be permeabilized or sectioned for introduction of the substrate. Thus, this conventional staining procedure generally results in the destruction of the stained cells. In light of this limitation, a secreted GUS would allow for development of non-destructive marker system, especially useful for agricultural field work.
The present invention provides gene and protein sequences of secreted &bgr;-glucuronide, variants thereof, and use of the protein as a transformation marker, while providing other related advantages.
SUMMARY OF THE INVENTION
In one aspect, an isolated nucleic acid molecule is provided comprising a nucleic acid sequence encoding a secreted form of &bgr;-glucuronidase, wherein the nucleic acid sequence comprises the amino acid sequence as presented in
FIG. 3
SEQ ID No: 2 or hybridizes under stringent conditions to the complement of the sequence comprising nucleotides 1662-3467 of
FIG. 1
SEQ ID No: 1 and which encodes a functional &bgr;-glucuronidase. In preferred embodiments, the nucleic acid molecule comprises nucleotides 1662-3467 of
FIG. 1
SEQ ID No: 1 or encodes the amino acid sequence of
FIG. 3
, SEQ ID No: 2 or a variant thereof.
In another aspect, the invention provides an isolated secreted form of &bgr;-glucuronidase, wherein &bgr;-glucuronidase is encoded by the isolated nucleic acid molecule or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of
FIG. 1
SEQ ID No: 1 and which encodes a functional &bgr;-glucuronidase. In a preferred embodiment, the isolated secreted form of &bgr;-glucuronidase comprises the amino acid sequence of
FIG. 3
, SEQ ID No: 2 or a variant thereof.
The invention also provides vectors and host cells, comprising a nucleic acid molecule encoding a secreted form of &bgr;-glucuronidase, wherein the &bgr;-glucuronidase sequence is in operative linkage with a promoter element. In preferred embodiments, the promoter element is a promoter derived from a plant pathogen. Preferred host cells are selected from the group consisting of a plant cell, an insect cell, a fungal cell, an animal cell and a bacterial cell.
The invention also provides a method of producing a secreted form of &bgr;-glucuronidase, comprising: (a) introducing a vector comprising a nucleic acid molecule encoding a microbial &bgr;-glucoronidase into a host cell, wherein the vector comprises nucleic acid sequence encoding the &bgr;-glucuronidase is expressed. The method may further comprise isolating the &bgr;-glucuronidase from cell supernatant or periplasm.
In other aspects, the invention provides methods of introducing a controller element into a host cell, monitoring expression of a gene of interest or a portion thereof in a host cell, monitoring activity of a controller element in a host cell, transforming a host cell with a gene of interest or portion thereof, and positive selection for a transformed cell.
In other aspects, transgenic cells are provided, such as plant cells, insect cells, and transgenic plants and insects.
In other aspects, kits comprising microbial GUS are provided.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.
REFERENCES:
patent: 5599670 (1997-02-01), Jeffereson
patent: 2197653 (1988-05-01), None
patent: 4023982 (1992-01-01), None
patent: 4267876 (1992-09-01), None
patent: 6256196 (1994-09-01), None
patent: 7274948 (1995-10-01), None
patent: WO89 03880 (1989-05-01), None
patent: WO96 37609 (1996-11-01), None
patent: WO99 13085 (1999-03-01), None
Cheon et al. Nodullin-24 follows a novel pathway for integration into the peribacteroid membrane in soybean root nodules. J. Biol. Chem. vol. 269(9):6598-6602 Mar. 1994.*
Farrell et al. Manipulation of beta -glucuronidase for use as a reporter in vacuolar targetting studies. Plant Mol. Biol. vol. 15:521-825 Jun. 1990.*
Chan et al. Novel gene expression system for plant cells gbased on induction of alpha-amylase promoter by carbohydrate starvation. J. Biol. Chem. vol. 269(26):17635-17641 Jul. 1994.*
Pang et al. Use of the signal peptide of Pisum viciln to translocate beta-glucuronidase in Nicotania tabacum. Gene. vol. 112:229-234 Apr. 1992.*
Iturriaga et al. Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco. The Plant Cell. vol. 1:381-390 Mar. 1989.*
Nelson KE et al.,Thermotoga maritimebeta-glucuronidase,Database PIR2 ‘Online’ EMBL,Heidelberg, Germany; ID/AC AE001766;
Harcourt Rebecca Louise
Jefferson Richard A.
Keese Paul Konrad
Kilian Andrzej
Wilson Katherine Joanna
Center for the Application of Molecular Biology to International
Foley & Lardner
McKelvey Terry
Sandals William
LandOfFree
Microbial &bgr;-glucuronidase genes, gene products and uses... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Microbial &bgr;-glucuronidase genes, gene products and uses..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Microbial &bgr;-glucuronidase genes, gene products and uses... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2828760