Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-09-11
2002-08-06
Prouty, Rebecca E. (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C435S091410, C435S243000, C435S320100, C536S024100, C536S024200, C536S023400, C536S023500
Reexamination Certificate
active
06429292
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to a repressor molecule for a glucuronidase operon and, more specifically, to amino acid and DNA sequences of a repressor and uses for a repressor protein.
BACKGROUND OF THE INVENTION
The natural habitat of
E. coli
is the gut, and the &bgr;-glucuronidase 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 perrnease (U.S. Pat. No. 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. This circulation of hydrophobic compounds resulting from the opposing processes of glucuronidation in the liver and deglucuronidation in the gut is termed enterohepatic circulation. This phenomenon is of great physiological importance because it means that, due in large part to the action of microbial &bgr;-glucuronidase, many compounds including endogenous steroid hormones and exogenously administered drugs are not eliminated from the body all at once. Rather, the levels of these compounds in the bloodstream oscillate due to this circulatory process. This process is of great significance in determining pharmaceutical dosages, and indeed some drugs are specifically administered as the glucuronide conjugate, relying on the action of &bgr;-glucuronidase to release the active aglycon (Draser and Hill, 1974).
&bgr;-glucuronidase is encoded by the gusA locus of
E. coli
(Novel and Novel,
Mol. Gen. Genet.
120:319-335, 1973). gusA (GUS) is one member of an operon, consisting of three protein-encoding genes. The second gene, gusB (PER), encodes a specific permease for &bgr;-glucuronidase. The third gene, gusC (MOP), encodes an outer membrane protein 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 not constitutively expressed in
E. coli
; rather, transcription of the operon is regulated by several factors. The primary mechanism of control is induction by glucuronide substrates. This regulation is due to the action of the product of the gusR (formerly uidR) gene which encodes the repressor. gusR was mapped by deletion mutation analysis to the same region of the chromosome as gusA, lying upstream of gusA. GusR repression of &bgr;-glucuronidase activity has been shown by Northern analysis to be mediated by transcriptional regulation: RNA from uninduced cultures of
E. coli
does not hybridize to a gusA probe, in contrast to the strong hybridization observed to RNA extracted from cultures that had been induced with methyl &bgr;-D-glucuronide (Jefferson, DNA Transformation of
Caenorhabditis elegans
: Development and Application of a New Gene Fusion System. Ph.D. Dissertation, University of Colorado, Boulder, Colo., 1985). Presumably, therefore, GusR represses gusA transcription by binding to gusA operator sequences, thereby preventing transcription. This repression would then be relieved when a glucuronide substrate binds to the repressor and inactivates it.
The present invention provides gene and protein sequences of glucuronide repressors and use of the repressor for controlling gene expression and detecting glucuronides, while providing other related advantages.
SUMMARY OF THE INVENTION
This invention generally provides isolated nucleic acid molecules encoding a glucuronide repressor. In particular, a nucleotide and amino acid sequence of the
E. coli
glucuronide repressor (gusR) are provided. In preferred embodiments, the nucleotide sequence of the repressor is presented in SEQ. ID. NO: 1 or a variant thereof. In certain embodiments, nucleic acid molecules that hybridize to gusR are provided. Nucleic acid sequences that encode glucuronide binding site of a glucuronide repressor are presented.
In another aspect, this invention provides a glucuronide repressor protein that binds to a glucuronide operator and that binds to a glucuronide, wherein the binding to the operator is inversely dependent on glucuronide binding. In certain preferred embodiments the repressor comprises the sequence presented in SEQ. ID NO: 2 or a variant thereof. In other preferred embodiments, the repressor comprises a fusion protein of a glucuronide binding site or domain and a nucleotide-binding domain.
In yet other aspects, methods for isolating a glucuronide are provided, comprising (a) contacting a glucuronide binding domain from a glucuronide with a sample containing a glucuronide, wherein the glucuronide binds to the repressor protein; and (b) eluting the glucuronide from the repressor.
Other aspects provide methods for determining the presence or detecting the presence of a glucuronide in a sample, comprising (a) binding a repressor protein to a nucleic acid molecule comprising a glucuronide operator sequence to form a complex; (b) contacting the complex with a sample containing a glucuronide, wherein the glucuronide binds to the repressor protein causing release of the protein from the nucleic acid molecule; and (c) detecting release of the protein.
In other aspects, methods are provided for controlling gene expression of a transgene, comprising (a) transfecting or transforming a cell with a nucleic acid molecule comprising a nucleotide sequence encoding the repressor protein, a glucuronide operator sequence, and a transgene, wherein the operator is operably linked to the transgene; and (b) contacting the cell with a glucuronide that binds to the repressor protein; wherein the glucuronide causes the repressor protein to release from the operator sequence, thereby allowing expression of the transgene.
In yet other aspects, methods are provided for identifying a vertebrate glucuronide transport protein, comprising doubly transfecting a host cell lacking transport activity with a reporter gene under control of a glucuronide repressor and an expression library constructed from vertebrate RNA, and screening for expression of the reporter gene in the presence of a glucuronide.
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 herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are incorporated by reference in their entirety.
REFERENCES:
patent: 5268463 (1993-12-01), Jefferson
patent: 5432081 (1995-07-01), Jefferson
Blanco C. et al. Cloning and endonuclease restriction analysis of uidA and uidR genes in E.coli K12: determination of transcription direction for the uidA gene. J. Bacteriol. Feb. 1982, vol. 149:587-594, Feb. 1982.*
Blanco C. et al. Negative dominant mutations of the uidR gene in E.coli:genetic proof for a cooperative regulation of uidA expression. Genetics. Feb. 1986, vol. 112: 173-182.*
Artandi SE et al. TFE3 contains two activation domains, one acidic and the other proline rich, that synergistically activate transcription. Nucleic Acids Res. 1995, vol. 23(19): 3865-3871, Feb. 1982.*
Jefferson R.A. Gen Bank Accession No. AAA68922, dated Jun. 27, 1995.*
Blanco et al., “Cloning and Endonuclease Restriction Analysis of uidA and uidR Genes inEscherichia coliK-12: Determination of Transcription Direction for the uidA Gene,”Journal of Bacteriology 149(2):587-594, 1982.
Blanco, “Transcriptional and translational signals of the uidA gene inEscherichia coliK12,”Mol Gen Genet 208:490-498, 1987.
Blanco et al., “Negative Dominant Mutations Of The uidR Gene InEscherichia Coli: Genetic Proof For A Cooperative Regulation Of uidA Expression,”Genetics 112: 173-182, 1986.
Ritzenthaler et al., “In
Jefferson Richard A.
Leader Michael
Wilson Katherine J.
Prouty Rebecca E.
Rao Manjunath N.
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