Positive Selection - Overview

This landscape covers use of a gene for selection that converts a neutral or toxic compound to a growth-promoting molecule.  See also technical information for one mechanism that can be improved cooperatively.

About this technology landscape

Authored by Wei Yang and Marie Connett Porceddu.  Assistance with some of the text and figures was provided by Shoko Okada and Leon Smith.  An attorney opinion on one of the critical path patents was provided to CAMBIA by Foley and Lardner.   We are grateful for technical assistance from Dr Nick dos Remedios and Neil Bacon for the web version and the development of software for biological sequence linking.  We also thank Heidi Loder for proofreading the whole document.  The preparation of this technology landscape was partially supported by a grant from Horticulture Australia Limited (Grant No.: HG03034).  We welcome updates and inputs by others through the comments interface available on every page of this version of the technology landscape.

The positive selection approach has been of interest to address some issues associated with currently used negative selection methods and selectable marker genes:

• In some areas, public acceptance questions use of certain selectable marker genes, notably genes encoding resistance to antibiotics, and some regulatory agencies discourage use of such systems in plants to be released into the field.
• Selection systems based on genes conferring resistance to antibiotics or herbicides ("negative selection") also stress living transgenic cells, in part by causing the release of phenolic substances from non-transformed, dying plant cells that may affect growth of transformed cells. This effect, which can hurt regeneration, limits their value in challenging tissue culture systems.

However, the use of both positive selection and certain negative selectable markers is constrained in certain jurisdictions by broad patents owned by companies that do not license them widely.  Patents and patent applications with claims covering positive selection are described in this landscape.

Chapter 1: Introduction

For genetic transformation of plants, when transformation frequencies are low, selectable markers are often used.  The expression of a selectable marker gene results in a product that allows the survival of the transformed cells in the presence of a selective agent that prevents regeneration of the non-transformed cells. 

Selectable markers mainly fall into two categories, according to the mode of action of the selectable gene product:  either conferring resistance to transformed cells by enzymatic detoxification of the selective agent,  or providing a growth advantage to transformed cells over non-transformed cells.  Selection methods based on the former mechanism are generally refered to as "negative selection", whereby the transformed cells are able to tolerate as substance which inhibits the growth of or even kills non-transformed cells.   These include selection based on antibiotics and herbicides, as described in details in the technology landscapes "Antibiotic resistance genes and their uses in genetic transformation, especially in plants" and "Resistance to Phosphinothricin".  Selection methods based on the latter mechanism are referred to as "positive selection", the subject of this technology landscape.

Rather than conferring resistance to a negative or toxic substance, positive selection involves conferring onto the transformed cell a metabolic advantage such as the capability of sugar consumption, or other competitive advantages for stimulating cell growth over nontransformed cells such as response to hormone and adaptation to extreme temperature.  Positive selection systems in the purest sense identify and select genetically transformed cells without damaging or killing the non-transformed cells in the population, and without co-introduction of antibiotic or herbicide resistance genes.

Claims define what is patented

It is the claims that define the boundaries of the patent owner's rights. Remember that the patent owner's rights are exclusionary: (s)he may exclude others from making, using, selling, offering to sell, and importing the patented invention (e.g. a product or a process), and from importing a product made by a process patented in the importing country. To determine if someone is infringing a patent, (i.e. making, using the invention, etc. without the patent owner's permission), the allegedly infringing product or process is compared only to the claims of that patent.

Claims cannot to be interpreted in a vacuum. Although claims define the invention, the scope of the claimed invention is not always clear from reading the plain language of the claim. Claim interpretation can be difficult; a proper analysis is done by reading the claims in the context of the specification and in the context of the "prosecution history" (the back and forth negotiations between the patent applicant and the patent office regarding the claim language), and no particular interpretation may be binding unless and until there is litigation.  Thus, although claims in this technology landscape were analyzed from the plain language and the specification, scope of the claimed inventions may not have always been precisely determined.

A patent application is not the same as a patent

In the countries where the patents analysed in this landscape were filed, patent specifications are published 18 months after the earliest filing. The publications contain the claims of the application as filed.  Often, the claims in the application are written much more broadly than is actually patentable. As the application is examined by a patent office and claim language negotiated, the claims may shrink in scope. The specification of a granted patent will usually be the same as when published, but it also may change as the result of a successful opposition or re-examination.

The truth about international patents

A patent is awarded by the government of a country and is valid only within its territorial boundaries. To obtain a patent that is valid in a particular country, a request must be made in that country's patent office.

Confusion and misunderstanding about "international patents" arises sometimes from the process of pursuing patents through the World Intellectual Property Organisation (WIPO). When looking at a WO ("World") published patent application, many people erroneously, but understandably, conclude that it is an application for a patent that will be valid in multiple countries. However, it is not a patent, and indeed it may never reach the national application phase or become a patent in any country. 

Through an international treaty (Paris Convention Treaty), a group of countries agreed to offer patent applicants in these countries a one-year period in which to file an application in one of the other countries without losing the benefit of their filing date. The advantage is that any "art" (related technology) that became known after the original filing date in the home country but before the filing date in another country could not be cited against the application. Thus, for example, an application filed in Canada could wait up to one year before filing the application in Mexico under the same "priority date". This would give an applicant time to decide whether the costs of filing in other countries is justified.

Later, a second treaty (Patent Cooperation Treaty, PCT) established another route to delay the additional filings in other countries. In this method, an international office was set up ((WIPO) to receive and process the applications. But now, the applicant has one year to file at the WIPO office, preserving rights and original filing date in those designated countries without having to go to the expense of actually filing in each country. Eventually to obtain a patent in these countries, the application does need to be filed in the national patent offices (the process is called "conversion"), pay fees, have translations done and comply with the regulations of each individual office. Depending on some procedural issues and fee payments, the applicant may have up to 30 months from the original filing date (the date the application was filed in the home country) to decide whether or not to file and undergo these expenses in each of these other countries.

What is "ownership" of a patent?

The legal owner of a patent is designated as the "Assignee" on United States patents and as the "Applicant" on patents in the rest of the world.

Patent law gives the patent owner the right to exclude others from making, using, offering for sale, selling, and importing the patented product and from using the patented process, as well as using, offering for sale, selling, or importing a product obtained directly from a patented process. These rights are tradeable. The typical form of trade is a license, in which some or all of the rights may be transferred. For example, the patent owner may license only some of the claims in a patent, all of the claims but only in a particular field of research, all of the rights but only in certain countries, or the right to make and use but not the right to sell.  The cost may vary from nil to high, and licenses may be exclusive or non-exclusive. 

The holder of an exclusive license can control the rights in much the same way as the "owner" of the patent.  However, unlike the ownership of a patent, which is a matter of public record, licenses in most countries can be private. Unless the parties to a license choose to reveal the relationship, it can be impossible to obtain information about it.

In this paper, the legal owner of record is noted. The cautionary note is that the legal owner may not be the party that is in control of the rights you want access to.

Why a technology landscape on positive selection systems?

In our experience, the intellectual property landscape in biotechnology areas is often not very well understood by the research community, especially the public sector. All too often rumours and misstatements about patents are passed along from researcher to researcher. This is an unfortunate situation; however, it is understandable as scientists are not generally familiar with reading and understanding patents.

With the increasing importance and emphasis on patents, it is becoming necessary for scientists to be versed in the field of intellectual property. To assist researchers and others in gaining an overview and understanding of relevant intellectual property, we are preparing a series of white papers in chosen topic areas of agricultural biotechnology.

As mentioned in the preface, positive selection systems used in plant biotechnology have certain advantages over negtive selection systems. Some of the positive selection systems have been applied to the transformation of not only  the model plants such as Arabidopsis, but also the crops of agricultural importance such as  maize, rice, wheat, patato, cassava, sugarbeet, orange and pearl millet. Many patents and patent applications concerning technology in the transformation of plant cells by positive selection of transformants hace been granted or filed.

With this paper and others now present on or planned for the Patent Lens, we strive to provide a readable and understandable overview of patents in some key areas of biotechnology. In this way, we hope to contribute to the public awareness of intellectual property issues that surround these key biotechnological tools. The information in the white papers is not exhaustive, but consists of selected documents found to broadly encompass the area. To satisfy the myriad questions and issues raised by the research or the interests of each person who visits this site would require a host of attorneys and an enormous amount of time. Instead, this paper is provided in order to open the door into the patent world and furnish platform knowledge from which additional self-directed investigation can be performed.

Because we believe that there is a great deal of value to tapping a broad knowledge base and collaborative problem-solving, we'd love to have your comments as we explore this landscape. Our comment interface allows you to weigh in.   We are expecially interested in your thoughts on prior art, information on the status of the patent (for example, whether it's expired, been abandoned or allowed to lapsed or if it's the subject of a litigation), and information about licensing (Is it currently licensed?  Exclusively? Non-exclusively?  Who's the licensing contact for the patent owner?) and other information or ideas you'd like to share about the technical subject matter of the patent.

The scope of the landscape

This technology landscape is mainly focused on positive selection methods based on enzymes for the metabolism of different carbon or nitrogen sources. However, we reckon that positive selection can have a broden meaning as long as a selection system is based on favouring transgenic cells while keeping the growth pattern  of the non-transgenic cells rather than killing the non-transgenic cells. These selection systems include:

• methods based on adaptation of temperature
• methods based on the interaction of a receptor with the target ligand
• methods for stimulating cell growth using polynucleotides encoding certain polypeptides (transcriptional activators)
• Hormone-dependent selection
• methods based on phosphorus utilization 

What is this technology landscape about?

This technology landscape on positive selection systems of plants presents basic scientific aspects, as well as the key intellectual property aspects, of the selection systems used in plant transformation that differentiate the transformed cells from the non-transformed cells by providing growth advantages to the transformed cells.

The Technology Overview section provides some historical perspective and basic scientific information about each paticular technology included in this landscape analysis. The IP Issues section comprises serach strategy, an overview on key patents and patent applications encompassing the technology and a table with detailed information on each patent and patent application including bibliographic data, independent claims and a summary of the claimed invention. Wherever possible, nucleic acid and peptide sequences claimed in the independent claims of granted patents from USPTO or some from EPO, and some of the PCT applications, are linked to the NCBI patent sequence database.

Examples on the analysis of patent claims for validity issues were given for certain patent families.

What is this technology landscape NOT about?

To learn more about patents and patentability, please visit our companion tutorial, "How to read a patent" and web sites such as the web site of the United States Patent Office and the web site of the World Intellectual Property Organization. Other resource sites may be found on the Links page.

The user should note that we do not analyze patents directed to methods of using growth inhibitory substances such as certain amino acids for selection. Some of these patents may dominate the agricultural patents discussed on this site. As well, we present only a selected set of patents and applications. The set represents what we consider to be key in the field. It is inevitable that others would have a different opinion about what is key and, as a result, may well have chosen a different set of patents.

Furthermore, the nature of the patenting systems worldwide means that new patents and patent applications may appear at anytime.  Similarly, patents may lapse or patent applications may expire or be replaced by new applications.  So, although we have tried to give the best coverage of the intellectual property surrounding positive selection systems, this landscape should not be viewed as a comprehensive coverage of the subject.  We would encourage those interested readers to offer comments on this work - with the view to improving the structure and content for all.

The patents discussed in the following chapters were identified using the following search strategies:

Chapter 2: Positive selection based on glucides

The general method of positive selection could use any growth-promoting catabolite precursor, and was first illustrated using various sugars that do not promote growth until altered by an enzyme for which the gene was the transformation marker.  The first patents were opposed by many parties on the basis that the idea had been described sufficiently prior to the patent filing that the grant of a monopoly to a different party was not justified, but after many appeals and some amendments these patents are still in force. 

Syngenta patent families on positive selection

Syngenta Participations AG owns two patent families (here named as A and B) on positive selection systems.  The assignments were previously to Novartis AG, one of the precursor companies that formed Syngenta (info), which had acquired certain patents from Danisco.

The patent family A, analysed in depth below, contains broad patents directed to a general method of selecting genetically transformed cells from a population of transformed and non-transformed cells by introducing a nucleotide sequence into plant cell so that the transformed cells have a competitive advantage in utilizing a compound; if covered by the claims, the introduced nucleotide sequence is not a marker gene for toxin, antibiotic or herbicide resistance, which are found in the "prior art".

The patent family B, analysed on a following page, is much less broad.  It is directed more specifically to a method of selecting genetically transformed cells from a population of transformed and non-transformed cells by transforming cells with a gene coding for an enzyme involved in mannose or xylose metabolism and selecting on a medium supplied with a compound that only the transformed cells are able to utilize. Such enzymes include xyloisomerases and phosphomanno-isomerases (such as mannose-6-phosphate isomerase and mannose-1-phosphate isomerase), phosphomanno mutase, mannose epimerases (those which convert carbohydrates to mannose or mannose to carbohydrates such as glucose or galactose); phosphatases (such as mannose-6-phosphatase and mannose-1-phosphatase), and permeases which are involved in the transport of mannose, or a derivative, or a precursor thereof into the cell.

Syngenta patent family A

This patent family has patents on positive selection granted in the United States, Europe, Canada, Australia and some other jurisdictions, as indicated in the patent information table at the bottom of this page.

Following Novartis' application for the European patent (EP 601092 B1) (and the US patent 5994629), oppositions were lodged in Europe by CAMBIA, BASF AG and Unilever PLC (opposition is not a process currently available in the United States).  Information that was presented in the European opposition for invalidating some of the claims in EP 601092 is provided here relative to the claims of US 5994629 (this example of validity analysis on patent claims was provided for CAMBIA by Foley and Lardner).

Validity analysis of certain claims of US 5994629

I. The Prosecution History of the Patent Family

A. Priority Claims and Patent Family Information

The US 5994629 patent is a continuation-in-part of Application No. 08/505, 302, filed on 3 October 1995, now U.S. Patent US 5767378. The US patents 5994629 and  5767378 each claim priority to GB 9304200 filed on 2 March 1993.

The US 5994629 patent is also a continuation-in-part of Application No. 08/378, 996, filed on 27 January 1995, now abandoned.  Application No. 08/378, 996, claims priority to application No. 08/196, 152, now abandoned, which was originally filed as application PCTIDK92/00252 on 27 August 1992. The US 5994629 patent, application No. 08/378, 996, application No. 08/196, 152 and PCT /DK92/00252 all claim priority to DK 1522/91, filed on 28 August 1991.  For the relationships between the applications, see diagram below.

B. Amendment History of the Claims of the US 5994629 Patent

According to the prosecution history of the US 5994629 patent, the applicants amended each of claims 1 and 7.  During prosecution in the USPTO, the applicants deleted "induces a positive effect" and "and" from claim 1.   Also in claim 1, the applicants added the limitations"...expression or transcription of...and...that only the transformed cells are able to utilize...".   In claim 7, the applicants deleted "a positive effect induced by" and added "...that only the transformed cells are able to utilize...".  In support of the amendments, the applicants argued, "claims 1, 7 and 26 are amended to clarify that the competitive advantage of transformed cells is due to the expression or transcription of the co-introduced nucleotide sequence.  Claims 1 and 7 are further amended to clarify that only transformed cells are able to utilize the 'supplied compound' by expression or transcription of the co-introduced nucleotide sequence and therefore have a competitive advantage."

The amendments to the claims are significant because the deletion of the limitation "induces a positive effect" broadens the scope of the claims such that the claims are open to negative effects.  However, the addition of the limitation "that only the transformed cells are able to utilize" narrows the scope of the claims by excluding compounds that may be used to a lesser extent by non-transformed cells, as compared to transformed cells.

C. References cited

During the prosecution of the US 5994629 patent, the applicants' attorney cited 3 references. The examiner cited no references and included a form PTO-892 with "NONE" written across its face.  None of the references from the parent applications of the US 5994629 patent or from the International Search Report or International Preliminary Examination Report of PCT /DK92/00252 were cited by either the examiner or the applicants in the prosecution of the US 5994629 patent.

II. Validity Analysis

A. Relevant Documents

The following prior art references are relevant to the validity analysis:

Doc1:  Jefferson, R. A. (1990), Gene Manipulation and Plant Improvement II, "New approaches for agricultural molecular biology: from single cells to field analysis," pp. 365-400., which qualifies under the provisions of 35 U.S.C. 102(b) as prior art because it described the invention claimed in the US 5994629 patent in a printed publication in 1990, more than one year prior to 27 August 1992, the earliest effective U.S. filing date for the US 5994629 patent.

Doc2:  Budar et al. (1986) "Introduction and expression of the octopine T-DNA oncogenes in tobacco plants and their progeny," Plant Science 46:195-206, which qualifies as prior art against the US 5994629 patent under the provisions of 35 U.S.C. 102(b) because it was published in 1986, more than one year prior to 27 August 1992.

Doc3: Jefferson R. A. (filed: 8 December 1989; earliest priority: 11 November 86) U.S. Patent 5268463 issued 7 December 1993, which qualifies as prior art against the US 5994629 patent under the provisions of 35 U.S.C. 102(e), because although issued after the earliest priority date for the US 5994629 patent, it was filed in the U.S. prior to 28 August 1991.

Doc4:  Sreekrishna et al. (filed 23 July 1986; earliest priority: February 1984) U.S. Patent No. 4857467 issued: 15 August 1989, which qualifies as prior art against the US 5994629 patent under the provisions of 35 U.S.C. 102(b) because it was published in 1989, more than one year prior to 27 August 1992.

Doc5:  Liijestroem et al. (14 October 1987) EPO 0241044 published application, which qualifies as prior art against the US 5994629 patent under the provisions of 35 U.S.C. 102(b) because it was published in 1987, more than one year prior to 27 August 1992.

Doc6:  von Schaewen et al. (1990) "Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photo synthesis and strongly influences growth and phenotype of transgenic tobacco plants," EMBO Journal 9:3033-3044, which qualifies as prior art under the provisions of 35 U.S.C. 102(b) because it described the invention in a printed publication in 1990, more than one year prior to 27 August 1992.

B. Summary of Analyzed Claims

For convenience, claim 1 of US 5994629 was broken into the following individual elements (the reason this is useful will be clear in the analysis of prior art, Section C below):

    [1.1] Genetically transformed plant cells comprising

    [1.2] a desired nucleotide sequence and

    [1.3] a co-introduced nucleotide sequence

    [1.4] wherein expression or transcription of the co-introduced nucleotide sequence in the transformed cells gives said transformed cells a competitive advantage

    [1.5] when a population of cells including the transformed and the non-transformed cells is supplied with a compound

    [1.6] that only the transformed cells are able to utilize, and

    [1.7] the desired nucleotide sequence codes for a gene other than a toxin, antibiotic or herbicide resistance gene.

Likewise, claim 7 was broken into the following individual elements:

    [7.1] A method of selecting genetically transformed cells from a population of cells comprising the steps of:

    [7.2] a) introducing into the genome of a plant cell a desired nucleotide sequence and

    [7.3] a co-introduced nucleotide sequence

    [7.4] wherein said desired nucleotide sequence or co-introduced nucleotide sequence codes for a sequence other than a toxin, antibiotic or herbicide resistance gene;

    [7.5] b) obtaining transformed cells;

    [7.6] c) supplying to the population of cells a compound

    [7.7] that only transformed cells are able to utilitize

    [7.8] wherein said transformed cells have a competitive advantage over non-transformed cells due to the expression or transcription of the desired nucleotide sequence or co-introduced nucleotide sequence in the       presence of the compound; and

    [7.9] d) selecting said transformed cells from the population of cells.

C.  Validity Analysis of Claim 1 under 35 U.S.C. 102

1. Claim 1 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by the Jefferson article (Doc1)

All of the elements of claim 1 are taught by the Jefferson article.

With regard to element 1.1, the Jefferson article discloses genetically transformed plant cells. Specifically, the Jefferson article discloses transformed tobacco plants. (p. 396, 1st full paragraph, lines 10-12).

With regard to elements 1.2 and 1.3, the Jefferson article discloses tobacco plants transformed with a CaMV 35S - GUS fusion. (p. 396, 1st full paragraph, lines 10-12) and further describes a gene fusion as "DNA constructions in which DNA sequences from two (or more) genes are combined." (p. 369, 2nd full paragraph, lines 1-4).

With regard to element 1.4, the Jefferson article discloses an experiment wherein the transformed cells have a competitive advantage over non-transformed cells. Specifically, the transformed cells which incorporate the GUS fusion can use tryptophyl-A-glucuronide as an auxin source, and the non-transformed cells cannot (p. 396, 1st full paragraph).  Since the transformed cells can use at least one more compound as an auxin source as compared to the non-transformed cells, the transformed cells have a competitive advantage over the non¬transformed cells.

With regard to element 1.5, the Jefferson article describes an experiment wherein non-transformed cells and transformed cells are supplied with tryptophyl-A-glucuronide (p. 396, 1st full paragraph).

With regard to element 1.6, the Jefferson article, in the same experiment states that tryptophyl-A-glucuronide "shows no auxin activity ... when assayed using untransformed cells," and indicates that the transformed cells "remained green and healthy," thus indicating that the transformed cells used the tryptophyl-A-glucuronide as an auxin source (p. 396, 1st full paragraph).

With regard to element 1.7, the Jefferson article discloses that the GUS gene "catalyzes hydrolysis of a very wide variety of A-glucuronides" and further discloses that "gene fusions are DNA constructions in which DNA sequences from two (or more) genes are combined such that the coding sequences of one gene (the responder)," e.g. here the GUS gene, "are transcribed and/or translated under the direction of another gene(s) (the controller)," i.e. here the CaMV 35S sequence (p. 371, 1st full paragraph, lines 5-6).   Thus, "the desired nucleotide sequence" directs transcription or translation of the GUS gene and does not code for a "toxin, antibiotic or herbicide resistance gene."

2. Claim 1 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by the Budar et al. article (Doc2)

All of the elements of claim 1 are taught by the Budar et al. article.

With regard to element 1.1, the Budaret al. article discloses the introduction of genes into tobacco cells by transformation and "normal transformed plants." (Abstract).

With regard to elements 1.2 and 1.3, the Budar et al. article discloses that "genes 1, 2, and 4. . . were cloned and introduced into tobacco cells by...leaf disk transformation." (Abstract)

With regard to element 1.4, the Budar et al. article discloses that the "product of genes 1 and 2 are involved in the production of the auxin ..." (p. 195, 2nd column, 1st full paragraph, lines 6-14).

With regard to elements 1.5 and 1.6, the Budar et al. article discloses that "our data show that genes 1 and 2 together ... can be used for positive and negative selections. One can select for the expression of gene 2 in plant cells if α-naphthalene acetamide is provided in the medium instead of an active auxin," (p. 205, 1st column, 4th full paragraph, lines 1-7).

Unless a cell population contained both transformed cells and non-transformed cells, there would be no need for selection. Further, if the non-transformed cells were able to use the α-naphthalene acetamide, there would be no way to select the transformed cells which, as noted, are able to use the α-naphthalene acetamide. Therefore, the disclosure of the Budar article sets forth providing a population of transformed and non-transformed cells with a compound that only the transformed cells are able to utilize.

With regard to element 1.7, the Budar et al. article discloses that "the protein encoded by gene 1 catalyzes the formation of indole-3-acetamide which is converted to IAA by the product of gene 2" (p. 195, 2nd col., 1st full paragraph, lines 6-14).  IAA, indole acetic acid, is an auxin (p. 195, 2nd full paragraph, lines 6-14).  "The product of gene 2 also catalyzes the formation of naphthalene acetic acid (NAA) when alpha-naphthalene acetamide is provided" (p. 195, 2nd col., 1st full paragraph, lines 6-14).

3. Claim 1 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by U.S. Patent 5268463 (Doc3)

All of the elements of claim 1 are taught by U.S. Patent 5268463.

With regard to element 1.1, U.S. Patent 5268463 discloses "plants transformed with a highly expressed CaMV 35S/GUS gene fusion." (col. 56, lines 2-3).

With regard to elements 1.2 and 1.3, U.S. Patent 5268463 discloses "a gene fusion comprising a GUS encoding nucleic acid under the control of a promoter/enhancer element, X, could be used to generate a transgenic plant. Tissue-specific activity of promoter X would be detectable by the observation that GUS activity was expressed in some plant tissue, but not others" (col. 20, lines 4-10).  Specifically U.S. Patent 5268463 discloses a CaMV 35S/GUS gene fusion, i.e. a desired nucleotide sequence and a co-introduced nucleotide sequence.

With regard to element 1.4, U.S. Patent 5268463 discloses an experiment in which "in the absence of auxin, leaf discs from control plants and CaMV 35S/GUS-transformed 'GUS plants' became chlorotic and died over a 7 week period (Fig. 18) ... on media in which tryptophyl glucuronide was the sole auxin source, only those leaves which expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide" (col. 56, lines 15-25).

With regard to element 1.5, U.S. Patent 5268463 discloses that "leaf discs from nontransformed and CaMV 35S/GUS transformed plants were exposed to media containing cytokinin and (i) no auxin, (ii) 1 μM tryptophyl-glucuronide, or (iv) 10 μM tryptophyl glucuronide." (co1. 56, lines 1-7). Thus, transformed and non-transformed cells were supplied with a compound.

With regard to element 1.6, U.S. Patent 5268463 discloses, in describing the outcome of the above mentioned experiment (iv) that "only those leaves that expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide" (coI. 56, lines 22-25).

With regard to element 1.7, U.S. Patent 5268463 discloses "a gene fusion comprising a GUS encoding nucleic acid under the control of a promoter/enhancer element, X, could be used to generate a transgenic plant.  Tissue-specific activity of promoter X would be detectable by the observation that GUS activity was expressed in some plant tissue, but not others" (col. 20, lines 4-10).  Specifically U.S. Patent 5268463 discloses a CaMV 35S/GUS gene fusion, e.g. a desired nucleotide sequence and a co-introduced nucleotide sequence. (col. 56, lines 1-4).  The CaMV 35S serves here as a promoter, (col 49, lines 6-7).

D. Validity Analysis of Claim 7 under 35 U.S.C. 102

1.  Claim 7 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by the Jefferson article (Doc1)

All of the elements of claim 7 are taught by the Jefferson article.

With regard to element 7.1, the Jefferson article describes an experiment wherein non-transformed cells and transformed cells are supplied with tryptophyl-A-glucuronide. (p. 396, 1st full paragraph). In the same experiment the Jefferson article discloses that tryptophyl-A-glucuronide "shows no auxin activity ... when assayed using untransformed cells," and indicates that the transformed cells "remained green and healthy," thus indicating that the transformed cells used the tryptophyl-A-glucuronide as an auxin source (p. 396, 1st full paragraph).  Since the transformed cells can use at least one more compound as an auxin source as compared to the non-transformed cells, the transformed cells have a competitive advantage over the non-transformed cells (p. 396. 1st full paragraph). The discussion of the experimental results concludes with the statement "other compounds are now being synthesized to achieve both negative and positive effects" (p. 396, 1st full paragraph).  Taken in the context of the discussion of "Fusion Genetics - Positive and Negative Selection for Gene Fusion Action" on p. 394, the term "negative and positive effects" on p. 396 clearly refers to negative and positive selection.

With regard to elements 7.2 and 7.3, the Jefferson article discloses tobacco plants transformed with a CaMV 35S - GUS fusion (p.396, 1st full paragraph, lines 10-12). The Jefferson article further describes a gene fusion as "DNA constructions in which DNA sequences from two (or more) genes are combined" (p. 369, 2nd full paragraph, lines 1-4).

With regard to element 7.4, the Jefferson article discloses that the GUS gene "catalyzes hydrolysis of a very wide variety of glucuronides" (p. 371, 1st full paragraph, lines 5-6). The Jefferson article further discloses that "gene fusions are DNA constructions in which DNA sequences from two (or more) genes are combined such that the coding sequences of one gene (the responder)," i.e. here the GUS gene, "are transcribed and/or translated under the direction of another gene(s) (the controller)," e. g. here the CaMV 358 gene.  Thus, neither the GUS gene nor the CaMV 35S gene code for a "toxin, antibiotic or herbicide resistance gene."

With regard to element 7.5, the Jefferson article discloses genetically transformed plant cells, specifically transformed tobacco plants (p. 396, 1st full paragraph, lines 10-12).

With regard to element 7.6, the Jefferson article describes an experiment wherein non-transformed cells and transformed cells are supplied with tryptophyl-A-glucuronide (p. 396, 1st full paragraph),

With regard to element 7.7, the Jefferson article, in the same experiment, states that tryptophyl-A-glucuronide "shows no auxin activity...when assayed using untransformed cells," and indicates that the transformed cells "remained green and healthy," thus indicating that the transformed cells used the tryptophyl-A-glucuronide as an auxin source (p. 396, 1st full paragraph).

With regard to element 7.8, the Jefferson article discloses an experiment wherein the transformed cells have a competitive advantage over non-transformed cells.  Specifically, the transformed cells which incorporate the GUS fusion can use tryptophyl-A-glucuronide as an auxin source, and the non-transformed cells cannot.  Since the transformed cells can use at least one more compound as an auxin source as compared to the non-transformed cells, the transformed cells have a competitive advantage over the non-transformed cells (p. 396, 1st full paragraph).

With regard to element 7.9, the Jefferson article describes an experiment wherein non-transformed cells and transformed cells are supplied with tryptophyl-A-glucuronide (p. 396, 1st full paragraph).  In the same experiment the Jefferson article discloses that tryptophyl-A-glucuronide "shows no auxin activity ... when assayed using untransformed cells," and indicates that the transformed cells "remained green and healthy," thus indicating that the transformed cells used the tryptophyl-A-glucuronide as an auxin source (p. 396, 1st full paragraph).  Since the transformed cells can use at least one more compound as an auxin source as compared to the non-transformed cells, the transformed cells have a competitive advantage over the non-transformed cells (p. 396, 1st full paragraph).  The discussion of the experimental results concludes with the statement "other compounds are now being synthesized to achieve both negative and positive effects" (p. 396, 1st full paragraph).  Taken in the context of the discussion of "Fusion Genetics - Positive and Negative Selection for Gene Fusion Action" on p. 394, the term "negative and positive effects" on p. 396 clearly refers to negative and positive selection.

2. Claim 7 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by the Budar et al. article (Doc2)

All of the elements of claim 7 are taught by the Budar et al. article.

With regard to element 7.1, the Budar et al. article discloses that "genes 1 and 2 together or gene 2 associated with α-naphthalene acetamide can be used for positive and negative selections. One can select for the expression of gene 2 in plant cells if α-naphthalene acetamide is provided in the medium instead of an active auxin." (p. 205, 1st col., 4th full paragraph, lines 1-7).

With regard to elements 7.2 and 7.3, the Budar et al. article discloses that "genes 1, 2, and 4 ... were cloned and introduced into tobacco cells by ... leaf disk transformation. " (Abstract).

With regard to element 7.4, the Budar et al. article discloses that "the protein encoded by gene 1 catalyzes the formation of indole-3-acetamide which is converted to IAA by the product of gene 2."  IAA, indole acetic acid, is an auxin.  "The product of gene 2 also catalyzes the formation of naphthalene acetic acid (NAA) when α-naphthalene acetamide is provided" (p. 195, 2nd col, 1st full paragraph, lines 6-14).

With regard to element 7.5, the Budar et al. article discloses the introduction of genes into tobacco cells by transformation and "normal transformed plants." (Abstract).

With regard to elements 7.6 and 7.7, the Budar et al. article discloses that "our data show that genes 1 and 2 together... can be used for positive and negative selections. One can select for the expression of gene 2 in plant cells if α-naphthalene acetamide is provided in the medium instead of an active auxin" (p. 205, 1st column, 4th full paragraph, lines 1-7).  Unless a cell population contained both transformed cells and non-transformed cells, there would be no need for selection.  Further, if the non-transformed cells were able to use the α-naphthalene acetamide, there would be no way to select the transformed cells which, as noted, are able to use the α-naphthalene acetamide.  Therefore, the disclosure of the Budar et al. article sets forth providing a population of transformed and non-transformed cells with a compound that only the transformed cells are able to utilize.

With regard to element 7.8, the Budar et al. article discloses that the "product of genes 1 and 2 are involved in the production of the auxin ..." (p. 195, 2nd col, 1st full paragraph, lines 6-14).

With regard to element 7.9, the Budar et al. article discloses that "genes 1 and 2 together or gene 2 associated with α-naphthalene acetamide can be used for positive and negative selections.  One can select for the expression of gene 2 in plant cells if α-naphthalene acetamide is provided in the medium instead of an active auxin" (p. 205, 1st col., 4th full paragraph, lines 1-7).

3. Claim 7 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by U.S. Patent 5268463 (Doc3)

All of the elements of claim 7 are taught by U.S. Patent 5268463.

With regard to element 7.1, U.S. Patent 5268463 discloses an experiment wherein "on media in which tryptophyl glucuronide was the sole auxin source, only those leaves which expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide" (col. 56, lines 20-26). U.S. Patent 5268463 further discloses that "to identify plants which express Y, one may identify plants that express GUS, as the expression of both genes is under the control of the same promoter" (col. 20, lines 44-50). Additionally U.S. Patent 5268463 discloses that "GUS gene fusions could be used to report on the expression of a second gene of interest" (col. 20, lines 16-19).

With regard to elements 7.2 and 7.3, U.S. Patent 5268463 discloses a gene fusion comprising a GUS encoding nucleic acid under the control of a promoter/enhancer element X, could be used to generate a transgenic plant.  Tissue-specific activity of promoter X would be detectable by the observation that GUS activity was expressed in some plant tissue, but not others" (col. 20, lines 4-10).  Specifically U.S. Patent 5268463 discloses a CaMV 35S/GUS gene fusion, i.e. a desired nucleotide sequence and a co-introduced nucleotide sequence.

With regard to element 7.4, U.S. Patent 5268463 discloses "a gene fusion comprising a GUS encoding nucleic acid under the control of a promoter/enhancer element X, could be used to generate a transgenic plant. Tissue-specific activity of promoter X would be detectable by the observation that GUS activity was expressed in some plant tissue, but not others" (col. 20, lines 4-10).  Specifically U.S. Patent 5268463 discloses a CaMV 35S/GUS gene fusion, e.g. a desired nucleotide sequence and a co-introduced nucleotide sequence (coI. 56, lines 1-4).  The CaMV 35S sequence serves here as a promoter (col. 49, lines 6-7).

With regard to element 7.5, U.S. Patent 5268463 discloses "plants transformed with a highly expressed CaMV 35S/GUS gene fusion" (col. 56, lines 2-3).

With regard to element 7.6, U.S. Patent 5268463 discloses that "leaf discs from nontransformed and CaMV 35S/GUS transformed plants were exposed to media containing cytokinin and (i) no auxin, (ii) 1 μM tryptophyl-glucuronide, or (iv) 10 μM tryptophyl glucuronide" (col. 56, lines 1-7).  Thus, transformed and non-transformed cells were supplied with a compound.

With regard to element 7.7, U.S. Patent 5268463 discloses, in describing the outcome of the above mentioned experiment (iv) that "only those leaves that expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide" (col. 56, lines 22-25).

With regard to element 7.8, U.S. Patent 5268463 discloses an experiment in which "in the absence of auxin, leaf discs from control plants and CaMV 35S/GUS-transformed 'GUS plants' became chlorotic and died over a 7 week period (Fig. 18)...on media in which tryptophyl glucuronide was the sole auxin source, only those leaves which expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide" (col. 56, lines 15-25).

With regard to element 7.9, U.S. Patent 5268463 discloses an experiment wherein "on media in which tryptophyl glucuronide was the sale auxin source, only those leaves which expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide" (col. 56, lines 20-26). U.S. Patent 5268463 further discloses that "to identify plants which express Y, one may identify plants that express GUS, as the expression of both genes is under the control of the same promoter" (col. 20, lines 44-50).  Additionally, U.S. Patent 5268463 discloses that "GUS gene fusions could be used to report on the expression of a second gene of interest" (col. 20, lines 16-19).

E.  Analysis of Dependent Claims 12, 21 and 22 under 35 U.S.C. 102

The validity of dependent claims 12, 21 and 22 of the US 5994629 patent is also analyzed here.  Each of these claims depends either directly or indirectly from claim 7 analysed above.

1. Dependent claims 12 and 22 of the US 5994629 patent fail to meet the requirements of 35 U.S.C. 102(b) because they are anticipated by the Jefferson article (Doc1)

All of the elements of claims 12 and 22 are taught by the Jefferson article.

With regard to dependent claim 12, the Jefferson article discloses that "the gus operon consists of the glucuronidase gene itself, encoding GUS (gusA - formerly uidA) ... ". (p. 391, 1st full paragraph, lines 1-2).  The Jefferson article further discloses "Table 1. GUS β-Glucuronidase ... Encoded by E. coli gusA (formerly uidA)" (p. 373, lines 1-2).

With regard to dependent claim 22, the Jefferson article discloses that "many other compounds are now being synthesized to achieve both positive and negative effects, for instance cyclohexamide-glucuronide, cytokinin glucuronide, etc." (p. 396, 1st full paragraph, lines 17-20).

2. Dependent claims 12, 21 and 22 of the US 5994629 patent fail to meet the requirements of 35 U.S.C. 102(b) because they are anticipated by U.S. Patent 5268463 (Doc3)

All of the elements of claims 12,21 and 22 are taught by U.S. Patent 5268463.

With regard to dependent claim 12, U.S. Patent 5268463 discloses transgenic plants expressing a β-glucuronidase gene fusion (col. 55, lines 60-63). U.S. Patent 5268463 further discloses that "the present invention relates to the β-glucuronidase (GUS) gene fusion...it is based on the surprising discovery that gene fusions comprising β-glucuronidase gene may be effectively expressed in a wide variety of organisms to produce active β-glucuronidase enzyme." (Abstract).

With regard to dependent claim 21, the Jefferson Patent discloses that "an additional and sometimes very useful technique is to use the specific β-glucuronidase inhibitor saccharolactone (Levvy, G. A., 1952, Biochem. J. 52:464) (Sigma S-0375, saccharic acid 1-4 latone, glucaric acid 1-4 lactone; glucarolactone) to corroborate the GUS-dependence of the fluorescence increase.  This inhibitor will eliminate glucuronidase activity at concentrations less than one millimolar, but the compound is unstable at neutral pH, so that care should be exercised during prolonged assays.  Because of this instability, it is preferable to use saccharolactone at up to 5 mM for assays up to half an hour.  Alternatively, the reaction and the inhibited reaction may preferably be performed at pH 6.0 or below.  GUS activity should not be affected by these conditions and saccharolactone is more stable at acid pH" (col. 31, lines 19-34).

With regard to dependent claim 22, U.S. Patent 5268463 discloses"...glucuronides comprising bioactive molecules can also be used as GUS substrates according to the invention; useful bioactive compounds include, but are not limited to steroid hormones non-steroid hormones and factors, lymphokines, auxins, cytokinins..." (col. 26, lines 40-47).

F.  Conclusion on the validity analysis

Based on the validity analysis, the folowing conclusion can be made:

A well-informed court should conclude that claims 1 and 7 of the US 5994629 patent are invalid because they fail to meet the requirements of 35 U.S.C. 102 in view of the disclosures of any one of the Jefferson article (Doc1), the Budar et al. article (Doc2), and U.S. Patent 5268463 (Doc3).

Furthermore, claims 12 and 22, which depend from to claim 7, are invalid as anticipated by either the Jefferson article or U.S. Patent 5268463.  Claim 21 is invalid as anticipated by U.S. Patent 5268463.

Further, without going into detail here, a well-informed court should hold that claims 1 and 7 are invalid because they fail to meet the requirements of 35 U.S.C. 103, based upon the disclosures of either the Sreekrishna et al. patent (Doc4) or the Liijestroem et al. European patent (Doc5) in combination with the von Schaewen et al. article (Doc6).

G.  Detailed patent information

Syngenta patent family B

This Syngenta patent family includes patents granted in United states, Europe, Canada and Australia on  positive selection system based on  mannose or xylose. The invention of this patent family is directed to a method for selecting genetically transformed plant cells comprising the seteps of providing plant cells with a gene coding for an enzyme involved in mannose or xylose metabolism and selecting the transformed cells with mannose or its derivative or precursor. The transformed plant cells are also claimed.

Technology overview

Mannose is a hexose sugar that can strongly inhibite seed germination, root growth and respiration of plants. The sugar can be taken up by roots and converted to mannose-6-phosphate by the action of hexokinase but can not be further utilized. The accumulation of mannose-6-phosphate inhibits phosphoglucose isomerase, causing a block in glycolysis. The production of mannose-6-phosphate also depletes the cell of inorganic phosphate (orthophosphate) that is required for ATP production. While mannose has no direct adverse effect on plants, as the toxicity is not mediated by the compound itself, growth inhibition is the consequence of its phosphorylation to mannose-6-phosphate by hexokinase.

Mannose

Phosphomannose isomerase (PMI, EC 5.3.1.8) is an enzyme that converts mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis that positively supports the growth of plant cell.  In 1984, a gene coding for phosphomannose isomerase (manA or pmi) was first isolated from Escherichia coli by Miles and Guest.  However, its first application in plants as a selectable marker gene was reported in 1998 by Joersbo et al.  The idea was that plant cells lacking PMI are incapable of surviving on synthetic medium containing mannose as a carbon source.  Introduction of  the manA (pmi) gene into plant cells enables those transformed cells to utilize mannose as a carbon source, improve the energy status and avoid accumulation of the derivatized mannose-6-phosphate, and thus gives the transformed cell the growth advantage over the non-transformed cells.

Fructose

To date, no endogenous PMI activity has been detected in plant cells, indicating that PMI selection may be useful in the transformation of many plant species.

Another selection system is based on xylose. Some plants such as potato, tobacco and tomato can not use D-xylose but can utilize D-xylulose as the sole carbon source.  However, a problem initially encountered when xylose was used in the selection medium was the induction of callus.  This proble was solved by addition of auxin inhibitor into the selection medium.

Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) catalyzes the isomerization of D-xylose to D-xylulose and the isomerization of glucose to fructose and is also termed as glucose isomerase. A gene (xylA) encoding xylose isomerase was reported to be isolated from Thermoanaerobacterium thermosulfurogenes or Streptomyces rubiginosus.

This system enables the effective selection of transgenic potato, tobacco and tomato cells using D-xylose as the selective agent.  Transgenic cells expressing the xylose isomerase gene can utilize xylose as a carbohydrate source and proliferate, whereas non-transgenic cells starve.

Specific patent information

Search strategy

Positive selection using glucuronide

CAMBIA holds patents in the United States and Australia that have claims on, among others, methods for selecting transformed cells based on the metabolism of various glucuronides that, when cleaved, could result in promotion of the growth of transformed cells.

Technology overview

GUS gene has been widely used as reporter gene for plant transformation since 1987. The enzyme coded by the GUS gene is β-glucuronidase, which hydrolyzes a wide variety of glucuronides. Therefore, the application of GUS gene can be extended to be used as a positively selectable marker. The utility of β-glucuronidase as selective marker relies on the fact that cells cannot grow on a β-glucuronide carbon source such as a glucuronide disaccharide unless β-glucuronidase is provided to cleave the β-glucuronide bond. The most useful example of such a disaccharide is cellobiuronic acid, which comprises β-glucuronic acid in [1-4] linkage to glucose. Only cells expressing β-glucuronidase can grow on a carbon source consisting only of cellobiuronic acid.

The positive selection system ba