AU620827B2 - A gene coding for glucuronide permease - Google Patents

Description

Declared e day of.. 19 Signed: R. Richard Anthony erson -f ri r I i

PCT

OPI DATE 23/05/89 AOJP DATE 29/06/89 APPLN- ID 26050 88 PCT NUMBER PCT/GB88/00936 INTERNATIONAL APPL -T) (51) International Patent Classification 4 (11) Internation b6atioJmI W 9/03880 C12N 15/00, C12P 21/02 Al (43) International Publication Date: 5 May 1989 (05.05.89) (21) International Application Number: PCT/GB88/00936 (22) International Filing Gate: 31 October 1988 (31.10.88) (31) Priority Application Number: 8725402 (32) Priority Date: (33) Priority Country: 29 October 1987 (29.10.87)

GB

(71)72) Applicant and Inventor: JEFFERSON, Richard, Anthony [US/GB]; 89 Norwich Street, Cambridge CB2 IND (GB).

(74) Agents: MATTHEWS, Heather, Clare et al.; Keith W.

Nash Co., Pearl Assurance House, 90-92 Regent Street, Cambridge CB2 IDP (GB).

(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FR (European patent), GB, GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), NO, SE (European patent).

Published With international search report.

Slhis u3cun:ent contains the amendme,.tst allowed Section 83 by the Superavi g Examiner onl and is correct for printing (54) Title: A GENE CODING FOR GLUCURONIDE PERMEASE (57) Abstract The present invention relates to t'le gene encoding the transport protein, glucuronide permease. Recombinant vectors encoding glucuronide permease can be used to transfect host cells. Expression of glucuronide permease by the transformants allow cellular uptake of p-glucuronides. This system permits the detection of p-glucuronidase activity in vivo, and can conveniently be used together with GUS gene fusions. In addition to providing an important complement to the GUS system, glucuronide permease also can be used in a method to selectively alter the permeability of cells. Because of the variety of substances which can be conjugated to p-glucuronides, glucuronide permease provides a cellular entrance for a multitude of compounds.

t i PCT/G G 88 00 9 3 b 2 1 Nc;-:mber 1938 C107. 2/ Title: Improvements in or relating to transport proteins 1. FIELD OF THE INVENTION The present invention relates to the transport protein glucuronide permease and ite use for selectively altering the permeability of live cells. This transport protein can be used to facilitate the cellular uptake of various compounds conjugated to (-glucuronides, including checmical markers and substances which alter cellular metabolism.

2. BACKGROUND OF THE INVENTION 2.1 O-GLUCURONIDES 0 -Glucuronides consist of glucuronic acid esters hai.g arious substituent groups (Figure In mammals, glucuronidation is a principle means of detoxifying or inactivatiing compounds, utilizing the glucuronyl transferase system. In humans, a number of hormones, including cortisoi anid idsosterone, certain antibiotics such as chloramphenicol, toxins such as dinitrophenol, and bilirubin are among the compounds which are conjugated to glucuronides by the glucuronyl transferase system and then excreted in urine or into the lower intestine in bile.

The bacterium Escheric lia coli has evolved to survive jin the mammalian intestine, and can utilize the excreted 0 -glucuronides as its sole carbon source. To do so, E.

coli has evolved mechanisms for the uptake and degradation of a wide variety of glucuronides, processes which are tightly linked genetically.

2.2 &-GLUCURONIDASE IS-Glucuronidase (GUS) is an enzyme which exhibits acid hydrolase activity, which cleaves the ester linkage between a glucuronide and its substituent. GUS has been characterized extensively in E. coli bacteria, as a very stable protein with a subunit weight of 68.2 KDa encoded by the uid A genetic locus. It catalyzes the cleavage of a wide variety of glucuronides, most of which are water soluble. As illustrated by Figure 1, a variety of 1q compounds, including,

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1 2but not-limited to, histochemical, fluorogenic or colorimetric markers can be conjugated to glucuronic acid to form the ester, p-glucuronide. These ,8-glucuronide chemical markers can be used as indicator substrates for the GUS enzyme.

Therefore, activity levels of GUS can be determined using sensitive colorimetric, fluorogenic, or histochemical detection methods.

Many organisms express little or no fl-glucuronidase, including higher plants such as potato and tobacco, the slime cerevisiae, and the fruitfly Drosophila melanogaster.

2.3. 8 -GLU.CUROINT.DAGE FROM E. COLT AS A GENE FJiN.E F0 Tne -glucuronidase structural gene was separated from, pro-moter/operator and Shine Delgarno, (ribosonme binding) sequences, fused with the E. ccli lac Z promoter sequence, and inserted into a plasmid vector by standard cloning tecniaes- The resulting construct, which places the 0-glucuronidase gene~ 4under the control of the la.c Z promoter, could be t-ranSfete 2into E. coli and resulted in high levels of f-glucuronidas.

expression (Jefferson et al., 1986, Proc. Natl. Acad. Sci.

U.S.A. 833;8447-S451 and see also UK specificarion No.

2197653).- If the lac Z promoter were active in initiating transcription, the A-qlucuroidase gene was excpressed.

To test whether expression of cloned p-glucuronidase could be controlled by other types of promoter sequences, the p-glucuronidase gene was coupled to various transcriptional promoters. Gene fusion between A-glucuronidase and either the Cauliflower mosiac virus (Ca)MV) 35S promoter or the ribulose bisphosphate carboxylase promoter were used to transform tobacco plants and achieved expression of 0-glucuronidase with what appeared to be promoter controlled tissue specificity (Jefferson et al., 1987, EMBO J. 6:3901-3907). Similar experiments, using mutants of the nematode Caenorhabitis A1~

SU~TUT~SHEET

1i o/CO 936 3 elegans which do not consitutively express p-glucuronidase, fused flanking regions of the col-l collagen gene or of a major sperm protein to the ,-glucuronidase gene, resulting in expression of a p-glucuronidase fusion protein; the data did not confirm tissue specificity or developmental control (Jefferson, et al., 1987, J. Mol. Biol. 193:41-46).

Importantly, the A-glucuronidase fusion protein showed functional enzymatic activity in organisms as diverse as bacteria, higher plants, and lower animals. Further, GUS activity could be detected in individual cells using histologic techniques.

These experiments used p-glucuronidase expression as an indicator of promoter activity. O-Glucuronidase served as a gene fusion marker, or "reporter gene", the expression of which was evaluated using a p-glucuronide indicator substrate.

In E. coli, the same genetic locus encodes pglucuronidase and transport proteins which facilitate the uptake of f-glucuronide substrates. The i'solated 6glucuronidase gene utilized in the gene fusion experiments described supra does not control the transport of 8glucuronide indicator substrates, so that measurements of 9glucuronidase activity were performed using tissue extracts or histologic sections.

2.4. GLUCURONIDE PERMEASE SThe phospholipid bilayer membrane has evolved to selectively retain molecules within cells, and prevent the promiscuous exchange of cellular contents with the extracellular milieu. Cells have, however, developed the capacity to transport selected compounds across the permeability barrier of the membrane, even against a concentration gradient. In general, without a specific transport mechanism, polar molecules are excluded from passing across a cell membrane. Because B-glucuronides are 35intrinsically polar molecules, bearing a negatively-charged -I 'c (L~is c n An -4ionized carboxyl group, these molecules are highly impermeant to cells, and require glucuronide permease for transport.

3. SUMMARY OF THE INVENTION The present invention relates to the gene encoding the transport protein, glucuronide permease. Recombinant vectors encoding glucuronide permease can be used to transfect host cells. Expression of glucuronide permease by the transformants allows cellular uptake of betaglucuronides. This system permits the detection of betaglucuronidase activity in vivo, and can conveniently be used together with GUS gene fusions.

.h In addition to providing an important complement to see the GUS system, glucuronide permease also can be used in a method to selectively alter the permeability of cells.

Because of the variety of substances which can be conjugated to beta-glucuronides, glucuronide permease provides a cellular entrance for a multitude of compounds.

In a first aspect the invention provides a recombinant DNA molecule comprising a glucuronide permease gene under the transcriptional control of a heterologous promoter, in which the glucuronide permease gene (i) comprises the sequence substantially as depicted in Figure 3 from about nucleotide number 106 to about nucleotide number 1464 or its functional eauivalent and (ii) encodes a molecule that has glucronide permease activity.

The invention also provides a host cell transformant containing a recombinant DNA molecule comprising a glucuronide permease gene under the transcriptional control of a herologous promoter, in which the gluruconide permease gene comprises the sequence substantially as depicted in Figure 3 from about nucleotide number 106 to 1r 1 4>NT 0 i;,:i~iu i 4a off& .00:: 0O 0000 0 0 o f 0000

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S S

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S. S *5S* about nucleotide number 1464 or its functional equivalent and (ii) encodes a molecule that has glucuronide permease activity.

The invention also includes within its scope a host cell transformant containing a recombinant beta glucuronidase gene and a recombinant DNA molecule comprising a glucuronide permease gene under the transcriptional control of a heterologous promoter, in which the glucuronide permease gene comprises the sequence substantially as depicted in Figure 3 from about nucleotide number 106 to about nucleotide number 1464 or its functional equivalent and (ii) encodes a molecule that has a glucuronide permease activity.

In further aspects the invention also provides a method of introducing a substrate for glucuronidase into cells of an organism and a method of altering the permeability of live cells of an organism, comprising introducing into the organism a recombinant DNA molecule in accordance with the first aspect of the invention.

4. DESCRIPTION OF THE FIGURES Figure 1 illustrates the reaction catalyzed by betaglucuronidase, and the structure of substrates transported by glucuronide permease.

Figure 2 illustrates the structure of various plasmids. The subcloning of the 3' end of pRAJ210, encoding the glucuronide permease, was done by cleaving pRAJ210 with Pst I (cleaving in the polylinker site of pUC9, proximal to the Xho I site of pRAJ210) and Nsi I which cleaves just 5' of the BamHI site of pRAJ220. The sequence of this region was determined by the method of Maxam and Gilbert.

Figure 3 illustrates the DNA sequence of the glucuronide permease gene on pRAJ 285. The sequence was determined as described in the legend to Figure 2. The sequence shown extends from the Nru I site within GUS (sequenced by dideoxy method) through the Nsi I site into the previously unsequenced region

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ni 0aVI 1 Ti 5 of pRAJ210. The sequence shown extends only just past the terminator codon of the permease. This is represented on the plasmid pRAJ285.. The rest of pRAJ210 has been sequenced, and is present on pRAJ280 pRAJ284.

FIG. 4 is a comparison of the amino acid sequences of glucuronide permease (top) and the melibiose permease (bottom) using the University of Wisconsin Genetics Computer Group compare programs. The lines between sequences indicate exact matches between the two sequences. Small gaps were introduced to maximize homology.

FIG. 5 is an analysis of the structure of the glucuronide permease using the University of Wisconsin Genetics Computer Group PepPlot program. The bottom panel shows the hydropathy plot generated using either the Goldman or the Kyte and Doolittle criteria. The many hydrophobic domains indicate potential alpha helical trans-membrane segments. This plot is very similar to a plot obtained analyzing the melibiose permease sequence.

5. DETAILED DESCRIPTION OF THE INVENTION The invention relates to the glucuronide permease gene, chimeric gene constructs and their expression products.

Recombinant nucleotide vectors constructed to contain the glucuronide permease gene can be used to transform a variety of host cells. When these constructs are engineered to contain appropriate expression control elements, transformants will express glucuronide permease or a derivative encoded by the glucuronide permease gene.

The invention is based, in part, upon the discovery that the expression of glucuronide permease in this fashion alters the permeability of the host cell membrane to glucuronides, thus permitting the entrance of 0-glucuronidase substrates. The method of the invention can provide for the detection of "reporter" p-glucuronidase in vivo, as well as for introducing metabolically active compounds into cells.

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For purposes of clarity in description, and not by way of limitation, the invention will be described in three parts: the glucuronide permease gene and expression product; (b) glucuronide permease chinieric genes and fusion proteins; and use of glucuronide permease and glucuronide permease fusion protein.

5.1. THE GLUCURONIDE PERMEASE GENE AND PROTEIN The glucuronide permease gene sequence and its deduced amino acid sequence are depicted in FIG. 3. These sequences, or their functional equivalents, can be used in accordance with the invention. For example, the sequences depicted in FIG. 3 can be altered by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to thu degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as depicted in FIG. 3 may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of the glucuronide permease sequence depicted in FIG. 3 which are altered by the substitution of different codons that encode the same or a functionally equivalent amino acid residue within the sequence, thus producing a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which 2 acts as a functional equivalent. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic and glutamic acid.

S- 2 2 1 November 1938 -7 The glucuronide permease sequence was derived as described in the subsections below.

5.1.1. SEQUENCING THE GLUCURONIDE PERMEASE GENE Upon sequencing the gene for f-glucuronidase (Jefferson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:8447-8451), sequence analysis indicated the presence of a second open reading frame of at least 340 bp, whose initiator codon overlapped the translational terminator of the A-glucuronidase gene. This open reading frame was found to be translationally active (Jefferson, 1985), Dissertation, University of Colorado, Boulder). Figure 2 illustrates the clones used to analyze the uid A locus of E. coli. pRAJ220 plasmid was used to deduce the sequence of p-glucuronidase; pRAJ210 was subcloned and the fragments 3' to the 0-glucuronidase gene, encoding the open reading frame, were sequenced. The resulting DNA sequence and predicted protein are shown in FIG.

3 5.1.2. LOCATING THE GLUCURONIDE PERMEASE LODING REGION By analogy with existing operons of E. coli, I proposed that the open reading frame encoded a permease protein that could facilitate the uptake of B-glucuronides. The lactose and melibiose operons (for example) consist of a gene encoding a hydrolytic enzyme followed by a cocistronic gene for the corresponding transport protein, or permease. This format,of. genes with interdependent functions being located on the same mRNA and subject to the same controlling mechanisms, is ubiquitous in bacteria. Because the substrates 3for glucuronidase are very polar, it is certain that they require active transport across the bacterial membrane. The level of genetic analysis performed on the uid locus would not have distinguished a mutation that eliminated p-glucuronidase function from a mutation that eliminated transport of a substrate, consistent with tight linkage between Pt, S Cru/B 936 1 0 glucuronidase and glucuronide permease. In 1961, Francois Stoeber, in his Ph.D thesis in Paris, France, described the properties of a glucuronide permease in E. coli. His work clearly established the existence of such a transport mechanism, but did not in any way address the genetics or molecular biology of the system. These facts led to the hypothesis that the open reading frame encoded the glucuronide permease. Further analysis has also indicated that the range of substrates for p-glucuronidase that can be transported by the glucuronide permease is much wider than that of any previously described glycoside permease.

5.1.3. ANALYSIS OF AMINO ACID SEQUENCE AND THE GLUCURONIDE PERMEASE PROTEIN The predicted amino acid sequence of the putative glucuronide permease was subjected to computer analysis to determine the existing sequences to which it had closest homology. The only two sequences that had significant homology to the glucuronide permease were the melB gene product, the melibiose permease, and the lacY gene product, the lactose permease. Of these, the homology with the melibiose permease is the strongest, and is shown in FIG. 4.

Interestingly, both the melibiose and lactose permeases are members of a class of sugar transporters that use the proton gradient of the cell membrane to drive the transport of the sugar against a concentration gradient. These permeases are also-in the unusual class that have been purified and shown to be active as single proteins, and whose activity can readily be reconstituted in vitro by addition of the pure permease to membrane vesicles.

The deduced amino acid sequence for glucuronide permease was subjected to a computer analysis to predict the structure of the protein. Such results are shown in FIG. The salient feature of the analysis is the extremely hydrophobic nature of the protein, and the long stretches of V9 5 -0 SU3.2Ti7UT2 SHEET 21 Novembcr 1988 -9 hydrophobic amino acids that could easily span a membrane.

The Kyte-Doolittle predictions of hydropathy (shown in the bottom frame) reveal hydrophobic regions that are located at almost identical positions to those of the melibiose permease, and at very similar positions to those of the lactose permease.

As explained in the following subsections, glucuronide permease may be readily produced using recombinant DNA techniques. However, in accordance with the invention, the production of glucuronide permease is not limited to genetic engineering techniques. All or portions of the amino acid sequence depicted in FIG. 3, including alterations such as substitutions, additions or deletions that yield functionally equivalent molecules, could be produced by chemical synthetic techniques well known to those skilled in the art.

5.1.4. MOLECULAR GENETIC DEMONSTRATION OF GLUCURONIDASE PERMEASE The proof of the glucuronide permease function was obtained by cloning the putative permease under the control of a heterologous promoter, in this case the promoter of the lactose operon of E. coli. When wild-type E. coli cells are planted oh LB agar petri plates containing the chromogenic substrate for GUS, 5-bromo-4-chloro-3-indolyl-beta-Dglucuronide (called X-Gluc), the colonies remain white. When excess glucuronidase is present in the cells, for instance when encoded by a plasmid, the colony turns blue, due to deposition of the indigo dye. The blue color in this case is caused by (GUS) enzyme that is released from the many broken cells in the colony. This tends to give a relatively diffuse blue colony, with dye being deposited on the agar around the colony as well as on the colony itself. If however, a plasmid containing, not GUS, but rather the permease gene linked to the lac promoter, is introduced into the wild-type cells, the colonies on X-gluc become deep blue. The phenotype is even 1 2 1 November 1989 more striking because the blue color is very discrete, and is strictly localized to the colony. These colonies do not produce any detectable GUS in the absence of X-gluc, but rather are induced by produce it when X-gluc is present. This is due to the X-gluc being transported into the cell, binding to the uidR gene product (the repressor of the uid operon) and allowing expression of GUS. This phenomenon requires the glucuronide permease action. This can best be seen in the series of cloning experiments summarized in Table 1.

The glucuronide permease gene was subcloned into pUC19 to give a gene fusion with lac that paused E. coli to give discrete blue colonies on X-gluc. This was then subjected to various changes to alter the reading frame of the predicted permease to determine whether the reading frame was required for the blue colony phenotype. Restriction endonuclease sites were chosen that were distributed throughout the gene. Some of these are indicated in FIG. 3. The restriction sites were cleaved and filled in to mutate the area around the site by shifting the putative reading frame. Such a shift occurring upstream of the glucuronide permease initiator codon (pRAJ218) showed no change in the color of the resulting colony.

However, frame shifts within the coding sequence eliminated or severely reduced the capacity of the gene to give rise to colored colonies. In particular the Nsi I site mutant (PRAJ282) was completely colorless and the Ace I site mutant (pRAI283) was almost completely colorless, with just a trace of blue after two days. The Ban II site frame-shift showed a faint trace of blue overnight with an obvious, but still quite pale, blue after two days on plates. The elimination of all blue color by the Nsi I mutant is expected, as the amount of permease made before the frame shift is very small on the order of 100 amino acids. The next frame shift, the Acc I mutant, showed a trace of permease action. This may be because the amount of permease made (more like half of the permease) could have residual activity. The Ban II mutant (in SUBSTITUTE SIEET N T 0

I

PCT/GB 8 CO 36 21 November 1988 11 which more than 80% of the permease is made) shows a definite but severely reduced activity. This is also as would be predicted, and demonstrates conclusively the role of the open reading frame in the development of the blue colony.

The deletion mutant that extended 3' from the Nco site at bp 1510 (pRAJ285) caused no obvious change, leaving dark blue colonies. This verified the 3' extent of the gene as predicted by DNA sequencing. The context of the start site of the gene was altered by oligonucleotide mutagenesis in'order to verify its location. This resulted in a permease gene deleted of sequences up to -12 from the initiator codon. This mutant showed an even darker blue colony (and smaller presumably due to the over-expression of the membrane protein). The higher level of permease in these cells may be due to better translation on the new mRNA, perhaps because of loss of attenuating sequences. This clone, pRAJ2S6, contains a Bam HI linker immediately 5' of the Shine/Delgarno sequence and ensures that the initiator codon is the first one presented on any hybrid mRNA produced from this cloned fragment. To make a more useful cassette, pRAJ286 was modified by the addition of another Bam HI linker at the 3' end. This vector, pRAJ287, contains the entire glucuronide permease gene as a Bam HI fragment within the polylinker sites of the plasmid pUC19.

Next, the ability of the glucuronide permease to transport substrates other than X-gluc was tested. If the permease could transport such a large heterocyclic molecule as X-gluc, it was reasonable that it could transport other complex glucuronides, and hence offer a general route to transporting GUS substrates. Two bacterial cultures, one containing the plasmid pRAJ230 and the other pRAJ210 (Jeffeson et al. 1986) were grown to similar densities in L broth. Both these cultures produce GUS within the cells. pRAJ210 also includes the DNA encoding the permease pRAJ230 is deleted of most of the permease gene. The cultures were washed "AL, ",i

AOL~-.

-12extensively to eliminate GUS from the medium, and incubated with a solution of 4-methyl-umbelliferyl glucuronide, a fluorogenic substrate for GUS. The culture containing PRAJ2i10 immediately began to fluoresce intensely, while the culture containing pRAJ21O did not. When the cultures were lysc d with a sonicator in the presence of fluorogenic substrate, both extracts showed intense fluorescence indicative of intact glucuronidase activity.

5.2. USES OF GLUCURONIDE PERMEASE AND GLUCURONIDASE FUSION PROTEINS The glucuroride permease gene may be used ia many different organisms to transport substrates for A~glicuronidase (GUS) into cells. Because this permease activity is encoded by a single polypeptide, and because there ~s no subsequent modification of the permease required for itsF inse'2.ioia into membranes or its function (by analogy with the melibiose and lactose permeases) it is reasonable to expect that expression of the perinease under the control of virtually Li any promoter in a transgenic organism will result in the 2transport of ,-glucuronides into the cells of that organism.

5.2.1. GLUCURONIDE PERMEASE USED TOGETHER WITH THE GUIS GENE FUSION MARKER In one embodiment of the p~resent invention, the glucuronide permease gene can be transfected together with GUS as part of the same construct, or incorporated into another vector cotransfector), such as a plasmid or a eukaryoticvector such as SV-40 (Mulligan and Berg, 1980, Science 209:1422-1427). This in turn will allow sub)strates for glucuronidase, including fluorogenic and colorimetric 8 glucuronide substrates, to be incorporated into live, undisru~pted cells, thus allowing detection of 0-glucuronic1ase ireporher gene activity in vivo, eliminatin;, th, constraints of TiY Z SHE~ET

XI

SPCT/U 8 CO9 3 6 2 1 Novmbzr 1988 13 tissue extracts and histologic procedures, and thereby providing for more general applicability of the GUS system.

5.2.2. GLUCURONIDE PERMEASE USED WITHOUT THE GUS GENE FUSION MARKER In another embodiment of the present invention, the glucuronide permease gene can be introduced into cells which have endogenous f-glucuronidase activity. By altering the number of glucuronide permease molecules present at the cell membrane, the permeability of the membrane to f-glucuronides can be controlled, and thus, glucuronide permease itself can function as a reporter gene.

5.2.3. GLUCURONIDE PERMEASE USED WITH A VARIETY OF PROMOTERS It has been shown, supra, that the glucuronide permease gene, as part of plasmid pRAJ210 and in its native position downstream from the B-glucuronidase gene, is actively transcribed and translated into a functional protein in bacteria. In other experiments, also descried supra, functional glucuronide permease was produced when the isolated Sglucuronide permease gene was controlled by the heterologcus lac promotor. Knowledge of the nucleotide sequence of the glucuronide permease gene, together with a knowledge of restriction enzyme specificities, allows one skilled in the art to combine the glucuronide permease gene with a variety of promotor elements and thus enable tight control of permease expression in cells transformed with the glucuronide permease gene. For example, promoters of different transcriptional activities could be used to produce corresponding levels of glucuronide permease; tissue-specific promoters, or controlled promoters, for example, are types of promoters which could be used.

Promoters which might be used to control, glucuronide permease expression provided for in the present invention include, but are not limited to, the SV40 early promoter f _0 A\rm. ii'' a wide variety of glucuronides, most of which are water soluble. As illustrated by Figure 1, a variety of A~AL compounds, including, PC T G63 C8C 0 9 3 14 region (Bernoist and Chambon, 1981, Nature 290,:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thy'midine kinase promoter (Wagner et al., 1981, Proc.

NatI. Acad. Sci. U.S.A. 78:144-1445), the regulatory sequences of the metallothionine gene (Brinster et al., 1982, NatureL 296:39-42); prokaryotic expression vectors such as the p lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl.

Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25)i see also 'Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase. promoter region (Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaic virus RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871), 4 and the chloroplast promoter for the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera- Estre! I et al., 1984, Nature 310:115-120); and the following animal transcript-'.onal control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoqlobulin gene control region which is active in lymph~oid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse 3mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel.

1:268-276), aipha-fetoprotein gene control region which is 3active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.

01T SUZ71 U T ZI ET A 4 L) To0 15 5:1639-1648; Hammner et al., 1987, Science 235:53-58); alpha I-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and DeVel. 1:161-171), beta.-globii gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the bran (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2-gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

5.2.4. GLUCURONIDE PERMEASE DELIVERED TO SELECTED CELL TARGETS Glucuronide perinease can be produced in large amounts by inserting the gene into an active expression vector anid allowing the gene to be expressed, for example, in bacteria.

In one embodiment of the pres~ent invention, the glucuronide permease could be chemically or genetically linked to a 2ligand, which could deliver the permease for insertion~ into cell membranes bearing the ligand receptor. By analogy to lactose and malibiose permease, the glucuronide permease.

should be able to integrate spontaneously into the cell membrane. For example, glucuronide permease could be coupled to the Fc region of an immunnoglobulin specific for a discrete popu~lation of mammalian cells. Upon binding to these cells, the antibody would deliver glucuronide permease for insertion into the cell membrane, thereby making a discrete popuiation of mammalian cells increasingly permeable to 6-glucuronides.

3In this example, selected ,A-glucuronides could then be used to various purposes (see infra), utilizing endogenous pglucuroniciase expressed by mammalian calls.

In another, related embodiment of the present invention, glucuronide permnease could be incorporated into -01,A4 IX7 intrinsically polar molecules, bearing a neyo u- ,4 4 16 PCTUi 8u/00936 j 2 1 Nov.aber 198 membrane vesicles, and thereby become inserted into the cell membrane. The membrane vesicles could contain various substances, including, but not limited to ,-glucuronide conjugated compounds.

5.2.5. GLUCURONIDE PERMEASE USED TO FACILITATE THE UPTAKE OF O-GLUCURONIDE CONJUGATES The present invention provides for the use of glucuronide permease in altering membrane permeability to Svarious compounds, utilizing -glucuronide conjugates and endogenous or exogenously-supplied P-glucuronidase actvity.

These compounds include, but are not limited to, the following substances which either may be conjugated to 8-glucuronide or transported themselves by glucuronide permease.

Indicator substances such as histochemical indicators including napthol and napthol ASB1; fluorogenic substances such as 4-methyl umbelliferone and fluoroscein 3- O-methylfluoroscein, and colorimetric indicators such as resorufin, p-nitrophenol, and phenolphthalein.

Catabolic substances such as cellobiuronic acid, a disaccharide which, when transported into the cell, is metabolized to glucose by 9-glucuronidase.

Growth factors, such as, the various peptide growth hormones, and in plants, cytokinin or auxin.

Toxic substances, such as snake venom toxins, including, according to their mode of action, cardiotoxins which cause irreversible depolarization of the cell membranes of heart muscles or nerve cells, neurotoxins which prevent neuromuscular transmission by blocking neurotransmitter receptors, and protease inhibitors which inhibit acetylcholine esterase and similar enzymes involved in nerve transmission.

Also included are phytotoxins such as ricin and abrin and bacterial toxins, herbicides such as dinitrophenol derivatives and chemotherapeutic agents.

SU- SiT -k V -*aEET u tc -J .1LLVRI CLuUUL IlUCJeuO1J.Ut2 IlulIUt L IUO Au 17 Various steroid hormones are excreted as conjugated A-glucuronides. Using glucuronide permease, these hormones could be recycled, and their mechanism of action potentiated in select cells. In another embodiment of the present invention, exogenous steroid-p-glucuronides could be targeted to glucuronide permease expressing cells.

Antibiotics which are deactivated by glucuronyl transferase, such as chloramphenicol, could be recycled, and their bioactivity half-life thereby extended.

facilitate the export of toxic glucuronides. For example, deposits from the nervous systems of infants born with erythroblastosis fetalis, a severe condition, caused by massive hemolysis secondary to maternal anti-Rh antibodies, which can result in hemoglobin deposition and bilirubin accumulation in the neonatal brain, with subsequent mental retardation.

The present invention is not to be limited in scope by the genes and proteins exemplified which are intended as but single illustrations of one aspect of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Plasmids containing the GUS gene have been deposited in November 1986 with the National Collection of Industrial and Marine Bacteria (NCIMB) Torry Research Station, PO Box 31,-135 Abbey Road, Aberdeen, UK AB9 8DG, including those plasmids.known as pBI101, including pBI101.1 (previously known as pTAKI), (NCIB accession No.

3) 12353), pBI101.2 (previously known as pTAK2) (NCIB accession No. 12354), and pBI101.3 (previously known as pTAK3) (NCIB accession No. 12355).

Sc ig a MI V deoitdinNvebr 96 ih h NtonlColcto o site into the previously unsequenced region ill I a- WO 89/03880 PCT/GB88/00936 18 Table 1.

Behaviour of E. coli strain DHS- containing various lacZ-glucuronide oermease fusions in ptJC19 Color on X-Gluc olates Plasmid pRAJ 280 pRAJ 281 pRAJ 282 pRAJ 283 pRAJ 284 pRAJ 285 pRAJ 286 oRAJ 287 (lacZ/permease fusion) (Sst I lacZ frameshift) (Nsi I frameshift) (Acc I fraieshift) (Sst I Ban II frameshift) (Nco I Pst I 3' deletion) end, Barn HI linker) end Barn HI linker) Blue Blue White White (trace of blue at 2 days) Whitish (very pale blue) Blue Dark Blue (small) Dark Blue (small)

I'

Claims (10)

1. A recombinant DNA molecule comprising a glucuronide permease gene under the transcriptional control of a heterologous promoter, in which the glucuronide permease gene comprises the sequence substantially as depicted in Figure 3 from about nucleotide number 106 to about nucleotide number 1464 or its functional equivalent and (ii) encodes a molecule that has glucuronide permease activity. S. 2. A recombinant DNA molecule according to claim 1, in I o oI°o which the glucuronide permease gene further comprises the nucleotide sequence substantially as depicted in Figure 3 nie from about nucleotide number 94 to about nucleotide number

4. Acnd105. 3. A host cell transformant containing a recombinant DMA molecule comprising a glucuronide permease gene under the transcriptional control of a heterologous promoter, in which the glucuronide permease gene comprises the sequence substantially as depicted in Figure 3 from about nucleotide number 106 to about nucleotide number 1464 or its functional equivalent and (ii) encodes a molecule that has glucuronide permease activity. S4. A host cell transformant according to claim 3, additionally contaning a glucuronidase gene. A host cell transformant containing a recombinant beta glucuronidase gene and a recombinant DNA molecule comprising a glucuronide permease gene under the transcriptional control of a heterologous promoter, in which the glucuronide permease gene comprises the J. O\ .ii tr m i it iucioa \-tvaen A/iy- u'i enc;e ,*ecl ha l lcrnd ;.nes ^ty sequence substantially as depicted in Figure 3 from about nucleotide number 106 to about nucleotide number 1464 or its functional equivalent and (ii) encodes a molecule that has glucuronide permease activity.

6. A host cell transformant according to claim 3, 4, or in which the glucuronide permease gene further comprises the nucleotide sequence substantially as depicted in Figure 3 from about nucleotide number 94 to about nucleotide number 105.

7. A host cell transformant according to claim 3, 4, S or 6, in which the host cell is an animal cell.

8. A host cell transformant according to claim 3, 4, or 6, in which the host cell is a plant cell.

9. A host cell transformant according to claim 3, 4, or 6, in which the host cell is a bacterium. A host cell transformant according to claim 3, 4, 5, or 6, in which the host cell is a fungus. j11. A host cell transformant according to claim 3, S4, 5, or 6, in which the host cell is a yeast.

12. A method of introducing a substrate for glucuronidase into an animal, plant, fungal, yeast or bacterial cell, comprising introducing into the cell a recombinant DNA molecule in accordance with claim 1 or 2.

13. A method of altering the permeability of a live animal, plant, fungal, yeast or bacterial cell, comprising introducing into the cell a recombinant DNA molecule in accordance with claim 1 or 2. \"?131 8A/435/5.12.91 tvr hydrophobic nature of the protein, ana Tne iony uii y s SU23T:P T SU1EET i: 9' I U i 21

14. A recombinant DNA molecule, substantially as herein defined, with reference to any one of Section 5.1.4 at pages 9 to 12 of the specification, Section 5.2.3 at pages 13 to 15 of the specification or Figure 2. A host cell transformant, substantially as herein described, with reference to Section 5.1.4 at pages 9 to 12 of the specification. DATED this 4th day of June 1991 RICHARD ANTHONY JEFFERSON By Their Patent Attorneys GRIFFITH HACK CO. I i Ui i 8713S/SLT/04.06.91 WO 89/03880 WO 8903880PCT/GB88/00936 1/6 ,8-glucuronidose reaction COOH 0 O-R KOH OH OH COO H 0OH KOH OH OH ROH ASSAYS histochemnical flu oro geni c R napthol nopthol ASHI R 4-methylumbelliferone R fluorescein R resorufin R p-nitrophenol R =phenolphthalein COlor Imet r ic Fig. SUBSTITUTE SHFFT I I 133HS fLnLus-sfls-- Eco RI [EcoRI FEcoRI FECO RI -BamHl *~mHl [BamHJl kBoHI Bcm HI .Eco RWY Hinc II Sou 3A Hinf I Eco R ,LBa mH I I 0 Xho I -Xhol -Xhol ini, I *Hincf Il .Sou 3A Sou 3A ~Sou 3A uC *Hae II -Sou3A'c Nru I Hinf I Hae LE Sou 3Al Sou 3A X- 0 LHind IIHindlI EcoRI I "T, 9 lZ 9S60fi88l9/1:3d O88COI68 OMX paqstpm oami sean~lno aqj o Ue esveuicd Dq ;o qsow S 1-y" SU~r703C"tiYUJVZ SHE-ET WO 89/03880 PCT/GB88/00936 3/6 E. coli Glucuronide Permease DNA Sequence of the Gene and Predicted Amino Acid Sequence 30 TCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAAAACGCTGGACTGGCATGAA 90 110 CTTCGGTGAAAAACCGCAGCAGGGAGGCAAACAATGAATCAACAACTCTCCTGGCGCACC Me tAsnG inC nLeuSerTrpArgThr 130 150 170 ATCGTCGGCTACAGCCTCGGTGACGTCGCCAATAACTTCGCCTTCGCAATGGGGGCGCTC IleValGlyTyrSerLeuGlyAspValAlaAsnAsnPheAlaPheAlaMetGlyAlaLeu 190 210 230 TTCCTGTTGAGTTACTACACCGACGTCGCTGGCGTCGGTGCCGCTGCGCGGGCACA TGCT PheLeuLeuSerTyrTyrThrAspValAlaGlyN/alGlyAlaAlaAlaArgAiaHisAla 250 270 290 GTTA CTGGTGCGGGTATTCGATGCCTTCGCCGACGTCTTTGCCGGACGAGTGGTGGACAG ValThrGiyAlaGlyI ieArgCysLeuArgArgArgLeuCysArgThrSerGlyGlyGin 310 330 350 TGTGAATACCGCTGGGGAAAATTCCGCCCGTTTTTACTC TTCGGTACTGCGCCGTTAATG CysGluTyrArcTrpGlyLysPheArgProPheLeuLeuPheGlyThrAlaProLeul-let 370 390 410 ATCTTCAGCGTGCTGGTATTCTGGG"TGCTGACCGACTGGAGCCATGGTAGCAAAGTGGTG IlePheSerValLeuValPheTrpValLeuThrAspTrpSerHisGiySerLysValVai Nsi 1 430 450 470 2 TATGCATATTTGACCTACIATGG7GCCTCGGGCTTTGCTACAGCCTGGTGAATATTCCTTAT TyrAlaTyrLeuThrTyrMetGlyLeuGlyf euCysTyrSerLeuValAsnIleProTyr 490 510 530 GGTTCACTTGCTACCGCGATGACCCAACAACCACAATCCCGCGCCr TCTGGGCGCGGCT GlySerLeuAlaThrAlaMe tThrGlnGlnProG inS erArgAiaArgLeuG iyAiaAia 550 570 590 CGTGGGATTGCC(CTTCATTGACCTTTGTCTGCCTGGCATTTCTGATAGGACCGAGCATT ArgGiyleAiaAiaSerLeuThrPhevalCysLeuAiaPheLeul leGlyProSerI le 610 630 Acc 1 650 AAGAACTCCAGCCCGGAAGAGATGGTGTCr-GGTA WCATTTCTGGACAATTGTGCTGGCG LysAsnSerSerPr'oGiuGiuMetVaiSerVali 1 rHisPheTrpThrleVaiLeuAia 670 690 710 ATTGCCGGAATGGTGCTTTACTTCATCTGCTTCAAAmCGACGCGTGAGAATGTGGTACGT IieAiaGiyMetVaiLeuTyrPheleCysPheLysSerThrArgGluAsnVaiVa!Arg 730 750 770 ATCGTTGCGCAGCCGTCATTGCAATATCAGTCTGCAAACCCTGAAACGGPLATCGCCCGCTGt IleVa iAiaGinProSerLeuAs nlleSerLeuGinThrLeuLysArgAsnArgProLeu Fi4. 3ai WO 89/03880 PCT/G B88/00936 416 790 810 830 TTTATGTTGTGCATCGGTGCGCTGTGTGTGCTGATTTCGACCTTTGCGGTCAGCGCCTCG PheMetLeuCyslleGlyAlaLeuCysValLeulleSerThrPheAlaValSerAlaSer 850 870 890 SerLeuPheTyrValArgTyrValLeuAsnAspThrGlyLeuPheThrValLeuValLeu 910 930 950 GTGCAAAACCCTGGTTCGTACTGTGGCATCGGCACCGCTGGTGCXXGGATGGTCGCGAGG ValGlnAsnProGlyTrpTyrCysGlyIle~lyThrAlaGlyAlaXxxMetValAlaArg 970 990 1010 ATCGGTAAAAAGAATACCTTCCTGATCGGCGCTTTGCTGGGi ACCTGCGGTTATCTGCTG IleGlyLysLysAsnThrPheLeuIlleGlyAlaLeuLeuGlyThrCysGlyTyrLeuLeu 1030 1050 1070 TTCTTCTGGGTTTCCGTCTGGTCACTGCCGGTGGCGTTGGTTGCGTTGGCCATCGCTTCA PhePh-eTrpValSerVa lTrpSerLeuProVa lAl. LeuValAlaLeuAlaI leAlaSer 1090 1110 1130 ATTGGTCAGGGCGTTACCATGACCGTGATGTGGGCGCTGGAAGCTGATACCGTAGAATAC I le GlyGlnGlyVa lThrMe tThrVa lMe tTrpAl aLe uG luAl aAs~pThrVa1G luTy r 15110Ban 11 1190 GGTGAATACCTGACCGGCGTGCGAATTGAAGGGCTCACCTATTCACTATTCTCATTTACC 1210 1230 1250 CGTAAATGCGGTCAGGCAATCGGAGGTTCAATTCCTGCCTTTATTTTGGGGTTAAGCGGA ArgLysCysGlyG lnAlaIleG lyGlySerIleProAlaPheI leLeuGlyLeuSerGly 1270 1290 1310 TATATCGCCAATCAGGTGCAAACGCCGGA.AGTTATTATGGGCATCCGCACATCAATTGCC Tyrl-leAlaAs nGlnValGlnThrProGluValIleMetGlyIleArgThrSerI leAla 1330 1350 1370 TTAGTACCTTGCGGATTTATGCTACTGGCATTCGTTATTATCTGGTTTTATCCGCTCACG LeuValProCysGlyPheMetLeuLeuAlaPheVallelleTrpPheTyrProLeuThr 1390 1410 1430 GAAAAATAAAACTGTAATGTACTAAATCGAC- AspLysLysPheLysGlulleValValGlulleAspAsnArgLysLysValGlnGlnGln 1450 1470 1490 TTACGGTTATATTTCAAATACGAACAGTCA LeulleSerAsplleThrAsnEnd Fig.3b SU526T'g-g UTE I V SUOSMklaU826.-ET L 714 WO 89/03880 PCT/GB88/00936 5/6 9 TIVGYSLGDVANNFAFAMGALFLLSYYTDVAGVGAAARAHAVTGAGI .RC 3 TKLSYGFGAFGKDFAIGIVYMYLMYYYTDVVGLSVGLVGTLFLVARIWDA 58 LRRRLCRTSGGQCEYRWGKFRPFLLFGTAPLMIFSVLVFWVLTDWSHGSK 53 INDPIMGWIVNATRSRWGKFKPWILIIGTLANSVILFLLF. SAHLFEGTTQ 108 VVYAYLTYMGLGLCYSLVNIPYGSLATAMTQQPQSRAkRLGAARGIAASLT II 1 1 1 1i I f I I I III 102 IV7FVCVTYILWGMTYTIMDIPFWSLVPTITLDKREREQLVPYPRFFASLA 158 15)2 FVCLAFLIGPS IKNSSPEEMVSVYHFWTIVLAIAGMVLYFICFKSTRENV GF.,vTA.GVTLPFVNYVGGGDRGFGFQMFTLVL. IAFFIVSTIITLRNVHEV 208 VRIVAQPSLNISLQTLKRNRPLFMLCIGALCVLISTFA.......... VSA 201 FSSDuNQPSaEGS:HLTLKAIVAIYKNLSC,:iLLGMAJAYNVASNIITGFA 249 SSLFYVRYVLNDTGLFTVLVLVQNPGWYCGIGTAGAXMVARIGKKNTFLI 251 I-YYFSYVIGDAD uLFPYYLSYAGAA.NLVTLVFFPIVLSRib-RILWAGASI 299 GALLGTCGYLLFFWVSVWSLPVALVALAIASIGQGVTMTVMWALEADTVE 3 01 LevLSC.GVLLLMALMSYHNWVhTVIAGILLNVGTA'LF~WVLQVIMVADIVD 349 YGEYLTGVRIEGLTYSLFSFTRKCGQAIGGS IPAFILGLSGYIANQVQ .T 351 YGEYKLHVRCESIAYSVQTMVVKGGSAFAAFFIAVVLGMIGYVPNVEQST 398 PEVIMGIRTSIALVPCGFMLLM'FVIIWFYPLTDKKFKEI 436 4~01 QALLGrAQFIMIALPTLFFMVTLILYFRFYRLNGDTLRRI 439 Fig.- 4 SUBSTITUTE SHEET, 57 52 107 101 157 151 207 200 248 250 298 300 348 350 397 400 *1 I; J7 <C PEPPLOT of, Gperm Pep ck: 52'41, 1 to 457 I i3j3J, pliJui.4 kprevIusIbly nlOUWiL aco f -s.n accession No. 12354), and pBI101.3 (previously known as pTAK3) (NCIB accession No. 12355). 3' Ju adi*Il INTERNATIONAL SEARCH REPORT International Application No PCT/GB 88/00936 I. CLASSIFICATION OF SUBJECT MATTER (if several classificatlon symbols apply, Indicate all) According to International Patent Classification (IPC) or to both Natlonal Classification and IPC IPC4: C 12 N 15/00, C 12 P 21/02 II. FIELDS SEARCHED Minimum Documentation Searched Classification System Classification Symbols IPC4 C 12 N, C 12 P Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched III. DOCUMENTS CONSIDERED TO BE RELEVANT' Category Citation of Document, with Indication, where appropriate, of the relevant passages i s Relevant to Claim No. is X Proc.Natl.Acad.Sci., 83, 1986 (USA) Richard A. 1-10 Jefferson et al: "P-Glucuronidase from Escherichia coli as a gene-fusion marker see page 8447 page 8451 see figure 2; the last 400 bases, p. 8450, line 25 p. 8451, line 3 X GB, A, 2197653 (RICHARD ANTHONY JEFFERSON) 1-10 May 1988, see figure 2C A The EMBO Journal, Vol. 6, No. 13, 1987 Richard A. 1-10 Jefferson et al: "GUS fusions:p-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. see page 3901 page 3907 Special categories of cited documents: 1o later document published after the international filing date document defining the general sae of the art which is not or priority date and not In conflict with the application but A cocumendeiningthe gneu r!lstn oh rhh ot cited to understand the p0lnciple or theory underlying the considered to be of particular relevance Invention earlier document but published on or after the International document of particular relevance; the claimed Invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or Involve an Inventive step which is cited to establish the publlcation date of another document of particular relevance;' the claimed Invention citation or other special reason (as specified) cannot be considered to Involve an Inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu. other means ments, such combination being obvious to a person skilled document published prior to the international filing date butn the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 17th January 1989 1 0 FEB 1989 International Searching Authority S f horld er EUROPEAN PATENT OFFICE e- -aDERPUTTEH Form PCTIISA/210 (second sheet) (January 1985) International Application No, PCT/GB 88/00936

111. DOCU MENT$ CONSIDERED TO 5E RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document. with indication, Where apPOtitS of the relevant passages Relevant to Claim No A J. Mol. Biol., Vol. 193, 1987 Richard A. Jefferson 1-10 et al: "Expression of Chimneric Genes in Caenorhabditis elegans. ",see page 41- page 46 t Form PCT1ISA1210 (extra sheest) (January 1905) I: ANNEX TO THE INTERNATIO-NAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. PCT/GB 88/00936 SA 24972 Thie annex lion~ the patent family memberm relating to the pntent docismcntq cited in thc attove-rocrntinned international -;rarch report. The miembers are aq contained in the European Patent Office 1lIP rile on 02/11/88 The Huropean Patent Office ir in no wa~y liahle for these pairticulars which nro merelY given for the pwipo-;c of informntion. Patent document PlIglicitio Patent family Piuhlication cited in search report p date member(-:) note GB-A- 2197653 25/05/88 NONE 0 w For more detail,; ahvrd this annex :see Oflficial Journal of the Feuropenn Pontent Office, No. 1 2192