Antibiotic Resistance Genes and their Uses in Plant Genetic Transformation - Overview

The production of transgenic organisms usually involves the delivery of a construct containing DNA of interest accompanied by a selectable marker gene, often an antibiotic resistance gene.  This enables the selective destruction of cells not containing the introduced DNA.  Selection is thought to be necessary for delivery techniques in which only a minor fraction of the treated cells become transgenic.

About this technology landscape

• This technology landscape was originally produced in 2003 by Carolina Roa−Rodríguez and Carol Nottenburg with funding from the Rockefeller Foundation.  Certain sections of this landscape were updated in 2006 by Dr. Marie Connett Porceddu (dates noted on the pages).  
• Updating has been necessary because there have been changes in patent status since that time; for example, on the USPTO database Patent Application Information Retrieval, which can be found at http://portal.uspto.gov/external/portal/pair, it can be seen that US Patent 6,255,560 has been allowed to lapse. Sometimes patent owners decide not to pay the annuity fees necessary to keep a patent in force. This patent claimed the use of a 35S promoter driving an antibiotic resistance gene, vectors containing such cassettes, and plants transformed with such cassettes. With the lapse of the patent, these cassettes and plants are no longer covered by valid claims of this patent. However, US Patent 6,174,724, whose claims dominate some of what was claimed in US Patent 6,255,560, is still in force. New patents may also have issued. If you notice that information in this landscape needs updating, help the community by posting in our Discussion Forum so that others will be aware of the information.
• Production by "The Team"

Antibiotic resistance as a selection system

General Aspects

Antibiotic resistance as a selection system

Plant biotechnology is based on the delivery, integration and expression of defined genes into plant cells, which can be grown to generate transformed plants. Efficiency of stable gene transfer is not high even in the most successful transfer systems and only a fraction of the cells exposed integrate the DNA construct into their genomes. Moreover, a successful gene transfer does not guarantee expression, even by using signals for the regulation of transgene expression. Therefore, systems to select the transformed cells, tissues or organisms from the non−transformed ones are indispensable to regenerate the truly genetically transformed organisms.

Antibiotic resistance genes allow transformed cells expressing them to be selected for out of populations of non−transformed cells. As part of this system, a selective toxic agent that interferes with the cellular metabolism is applied to a population of putatively transformed cells. The population of cells that has been transformed with and expresses a resistance gene is able to neutralize the toxic effect of the selective agent, either by detoxification of the antibiotic through enzymatic modification or by evasion of the antibiotic through alteration of the target.

The antibiotic resistance genes can be the genes of interest in their own right or they can be operatively linked to other genes to be transformed into the organisms.

Main components of an antibiotic resistance system

The effectiveness of a particular antibiotic resistance system depends mainly on the following elements:[add a comment]

• The selective agent. It should fully inhibit growth of untransformed cells. The lowest concentration of the toxic compound that suppresses growth of the non−transformed cells and does not cause detrimental effects to the transformed ones is used.
• The resistance gene. Transcriptional and translational control signals fused to the resistance gene determine to a great extent the expression level of resistance. In addition, the gene sequence plays an important role as some are more compatible with animal or plant systems or subgroups of animals and plants, such as monocotyledonous or dicotyledonous plants.
• The material to be selected. In the case of plants, sensitivity to the selective agent depends on many factors, including the explant type, the developmental stage, tissue culture conditions and the genotype.

Apart from these factors, for an antibiotic resistance system to be efficient and useful the selectable marker gene should be expressible in a wide variety of cells and tissues, the background metabolic activity or resistance should be minimal or negligible, and a clear phenotypic change should be visible.[add a comment]

The most popular antibiotic resistance marker genes

Among the most widely used antibiotic resistance genes as selectable markers are neomycin phosphotransferase II (nptII) and hygromycin phosphotransferase (hpt). There are also other marker genes like gentamycin acetyltransferase (accC3) resistance and bleomycin and phleomycin resistance, but these are not as commonly used.

The enzyme NPTII inactivates by phosphorylation a number of aminoglycoside antibiotics such as kanamycin, neomycin, geneticin (or G418) and paromomycin. Of these, G418 is routinely used for selection of transformed mammalian cells. The other three are used in a diverse range of plant species, however, kanamycin has proved to be ineffective to select legumes and gramineae.

Hygromycin phosphotransferase is a suitable marker system for both plant and animal systems. The HPT enzyme inactivates the antibiotic hygromycin B. Hygromycin is usually more toxic than kanamycin and kills sensitive cells more quickly. It is nowadays one of the preferred antibiotic resistance marker systems for transformation of monocotyledonous plants, particularly gramineae (cereals and forages).

Current issues on the use of antibiotic resistance genes

Part of the worldwide debate about genetically−modified organisms focuses on the safety of antibiotic resistance gene markers in crops destined for human and animal consumption. Diverse and, in some cases, contradictory opinions have been voiced. At times, the debate has been unhelpful and polarized. One of the problems is that despite an increasing body of scientific knowledge about genetic modification, the opinions put forward have often been based on perception and emotion, rather than scientific rationale.[add a comment]

In response to these concerns, scientists have focused efforts on identifying potential risks to the ecology, resistance management and food biosafety. The results should allow more informed decisions to be made with respect to this technology.[add a comment]

Engineered antibiotic resistance genes in products for consumption

Several commercial transgenic crops in development or production contain antibiotic resistance genes as part of their new genetic make−up. For instance, crops contain genes whose products confer resistance to kanamycin (the nptII gene), spectinomycin and streptomycin (the aad gene), and ampicillin (the bla gene). Concerns have been raised about whether this may lead to an increase in the occurrence of microbial populations resistant to antibiotics, thereby posing a risk to animal and human health.[add a comment]

Most antibiotic resistance genes used in biotechnology were originally isolated from bacteria. To be used in plants these genes undergo a series of modifications: regulatory elements in the DNA sequence are exchanged for those used in plant cells, and usually the gene sequence is also altered to reflect the preferred codon usage of plants. This would make horizontal gene transfer back to bacteria unlikely.[add a comment]

For further information, a list of recent scientific papers discussing the potential of horizontal transfer of genes from plants to bacteria present in the gut of humans or animals and other related issues has been compiled. The list is neither exhaustive nor comprehensive, but is representative of the research in this area.[add a comment]

Alternative methods to antibiotic resistance marker genes

Researchers have devised selection methods that avoid the use of antibiotic or herbicide resistance genes or eliminate them in the final transgenic product. The development of these methods are in part in response to the concerns about horizontal transfer of antibiotic resistance genes, the public perception of risk and the consumer acceptance of the marketed products. Another motivation has been the need for multiple selectable marker genes because the use of a selectable marker gene in a particular line precludes further use of the same selectable gene in subsequent transformations of the same line. The generation of a cultivar with several distinct desirable traits may require repeated transformations events, which would require the use of a different selectable marker for each transformation event. The number of suitable, multiple selectable markers available is limited at present. In addition, the presence of multiple homologous sequences in the same genome may cause instability of the transgenes.[add a comment]

Two general strategies have been pursued to avoid the use of antibiotic resistance genes:[add a comment]

1. elimination of the selectable marker gene in the resultant transgenic organism and
2. use of a non−toxic compound that favors or promotes the regeneration and growth of transformed cells expressing a transgene product that acts on the compound (positive selection).

In the first strategy, the methods currently employed are: [add a comment]

• Co−transformation. Two separate DNA constructs, one containing a gene of interest and the other having a selectable marker gene are co−transformed into the target cell. As the transformed genes are physically separated, the selectable marker can be eliminated by a variety of mechanisms after assessing the integration and expression of the gene of interest. The system depends on a high efficiency of co−transformation and on the integration of the co−transformed DNAs in distant loci. Co−transformation can be achieved by using either two separate plasmid molecules with the different DNAs in the same transformation agent, e.g. one Agrobacterium strain, or by using two different bacterial strains, each containing one of the two constructs.
  
• Site−specific recombination systems. These systems require an enzyme that acts in trans (it does not need to be operatively linked to the molecule upon which it acts) to catalyze recombination between two short, cognate DNA sequences. The marker gene, which is cloned between the short cognate sequences, is eliminated when the enzyme, a recombinase, acts upon the sequences. Some of the site−specific recombination systems used are FRT(asymmetric inverted repeat sequences)/FLP (specific recombinase) from Saccharomyces cerevisiae and loxP/Cre (causing recombination) from bacteriophage P1.
• Intra−genomic relocation of transgenes via transposable elements. Transposition of the maize element families Ac/Ds and the Spm/dSpm is a cut and paste mechanism that results in the excision of the elements of one locus prior to the reinsertion into a second locus. In this way, the selectable marker is removed via transposition of the transposable element that is flanking the marker gene. Failure of the transposable element to reintegrate will cause the selectable marker to be lost.
  

The second strategy, known as positive selection, uses selective, non−toxic compounds to exploit the auxotrophies of the transformed material, i.e., the material is unable to regenerate and grow in the absence of an external supply of specific compounds. These methods of positive selection are based on complementing the transformed cells with a gene(s) that enables them[add a comment]

• to produce the essential substance themselves, or
• to be able to utilize a substrate, which confers a metabolic advantage over the non−transformed cells.

Examples of positive selection systems are a[add a comment]

• transgenic glucuronidase in combination with the use of cytokinin glucuronides or other phytohormone glucuronides, and
• transgenes such as phosphomannose isomerase and xylose isomerase that confer the ability to metabolize alternative carbohydrate sources such as mannose and xylose.
  

Patents on antibiotic resistance genes

Overview

Despite the existence of alternative methods for selection of transgenic organisms, antibiotic resistance genes are still widely used as selectable markers because they are highly efficient, economical and straightforward. Therefore, they are still considered a very valuable tool at experimental and commercial levels. As with many enabling technologies, antibiotic resistance genes are proprietary technologies in the hands of a few entities.[add a comment]

The scope of patent protection ranges from the very broadly claimed use of any antibiotic resistance gene in plant transformation to the more restrictive use of particular antibiotic resistance systems in conjunction with particular promoters and selective agents.[add a comment]

The present paper analyses the extent of patent protection on[add a comment]

• the use of any antibiotic resistance gene, mainly for plant transformation; and
• the most commonly used antibiotic resistance marker genes: neomycin phosphotransferase II (nptII) and hygromycin phosphotransferase (hpt).

The analysis concentrates on the following aspects[add a comment]

• the entities that have been granted patents or have filed applications in the area;
• the geographical coverage of the patents;
• the nature of the inventions, whether they are products or processes or both, their components and limits;
• comparison between the protected inventions noting overlaps and differences; and
• some implications of dominant patents in this area.

The topics of analysis are Antibiotic resistance genes in general;[add a comment]

• Neomycin phosphotransferase gene;
• Hygromycin phosphotransferase gene;
• Dominant patents on antibiotic resistance genes.

Antibiotic resistance genes in general

IP aspects

Monsanto Company holds patent rights on the use of any antibiotic resistance gene as a selectable marker for plant transformation. Importantly, these proprietary rights apply only in the United States and are covered by three granted patents:

• US 5034322
• US 6174724
• US 6255560

These three United States patents are related to three other United States patents and one European patent. However, the other patents are directed to the more specific subject matter of chimeric genes for plant transformation containing the 35S Cauliflower Mosaic Virus (CaMV) promoter or the promoter of the ribulose−1,5−bis−phosphate carboxylase small subunit (rbcS) gene in combination with the neomycin phosphotransferase (npt) gene as the antibiotic resistance gene.

These patents directed to promoters are analysed in the technology landscape Promoters.

Bibliographic data

Antibiotic resistance genes in general
Patents granted to Monsanto

Actual granted independent claims

The protected inventions

Common features

The independent claims of all three United States patents are product claims. There are no method claims.[add a comment]

The product commonly claimed in all three patents is a chimeric construct comprising any antibiotic resistance gene under control of a promoter that works in plants. More specifically, the chimeric genes comprise:[add a comment]

• a promoter region expressible in plant cells,
• a structural DNA sequence encoding a polypeptide that confers antibiotic resistance to the plant cell, and a non−translated region encoding a mRNA polyadenylation signal.
  

Different Features

The independent claims of the United States patents differ from each other in the following aspects:

Analysis of the protected inventions

Definition of terms

Although most of the terms employed throughout the disclosure are clear and correspond to the common usage of the terms in science, the term promoter " naturally expressed" in plants is not expressly defined and, as such, leads to uncertainty about what promoters are covered by these claims. Does "naturally expressed" encompass only promoters from plant genes? or does it include promoters not found as part of plant genes but fully operable in plant cells? The following discussion provides a framework for approaching this issue.

What is a promoter "naturally expressed" in plants?

The inventors do not provide a precise definition for a promoter "naturally expressed in plants" in the disclosure. The file history of the United States patent 6174724, containing all the correspondence held between the U.S. Patent Office and the applicants during the examination process of the application until its issuance, does not reveal the exact scope of the concept either. However, a "guesstimate" of the concept can be drawn from the examples provided in the application and the statements made by the applicants and the examiner during the examination process.

During examination the patent application was initially rejected on the grounds of being enabling only for the use of the exemplified promoters from the nopaline synthase gene (nos) of the Agrobacterium Ti plasmid and from the ribulose−1,5−bis−phosphate carboxylase small subunit (rbcS) gene, a plant gene. At the time of the invention, 1983, identification, isolation and evaluation of promoters was not a routine task, and the examiner considered that the disclosure did not provide enough guidance for the use of any non−exemplified promoter expressed in plant cells. It appears that the examiner was envisioning promoters expressed in plants from any plant−expressed gene. Although the nos promoter is a promoter of bacterial origin, it was deemed to fall within the concept of plant−expressible promoters as it becomes operational only when the Agrobacterium T−DNA region integrates into the plant cell chromosome and commands the plant cell to initiate the transcription of the nos gene. The applicants finally overcame this ground of rejection by stating that full identification of the regulatory regions of a gene was not an absolute requirement for using the invention. They argued that they had provided a comprehensive method to evaluate the ability of non−exemplified promoters to drive the expression of an antibiotic resistance gene. Notably, the applicants did not disagree with the examiner's "definition" of naturally expressed promoters.

A further useful insight on the meaning of the term can be gleaned from the applicants' assertion that animal, yeast and bacterial−derived promoters are not plant−expressible promoters and therefore, are not expected to work in plants. They emphasized during the examination process that the promoter should be a plant−expressible promoter capable of functioning in a selected plant cell. The applicants made this assertion in light of experiments conducted by Herrera−Estrella et al. (Nature 303: 209−213, 1983) and Barton et al. (Cell 32: 1033−1043, 1983) on promoters from plant origin and non−plant origin driving heterologous genes in plant cells ¹. The exact quote by the applicants in the examination proceedings is as follows:

Therefore, a likely interpretation is that:

Limitations and implications of granted patents

Geographical limits

• It is worth emphasizing that the geographical scope of protection for the patented chimeric genes and other features of the inventions is confined to the United States and its territories. Monsanto may enforce its rights over these inventions only in the United States. Users in all other countries are free to employ in any way the products claimed in the patents. The only restriction would come when such users seek to import products, i.e. plants containing chimeric constructs claimed in the patents, into the United States. In this case, users without permission from Monsanto would be in breach of the rights of the patent holder.

Any antibiotic resistance and certain promoters as part of the constructs

• A basic chimeric gene, which is the subject matter of the claims of patents US 5034322 and US 6174724, corresponds to a chimeric gene similar to those routinely and widely used in plant transformation technologies all over the world. The comprising elements of the chimeric gene of the inventions are the essential components in a chimeric construct designed for the selection of transformed plants based on their acquisition of antibiotic resistance capacity. Thus, essentially, in the United States the use of any antibiotic resistance gene for transformation of plants is covered by the present Monsanto patents.
• The claims, however broad, do have certain limitations. The antibiotic resistance gene must be used in conjunction with a promoter derived from the opine synthesis genes of A. tumefaciens or from a rbcS gene ('532 patent) or a promoter naturally expressed in plants ('724 patent) ( i.e. promoter from a plant gene or a promoter from a non-plant gene that is normally expressed in a plant cell environment). These limitations potentially leave the window open for the use of promoters from genes of organisms such as bacteria (except maybe Agrobacterium/Rhizobium), fungi, animals and viruses which are not normally expressed in the plant cells. The inventors themselves ruled out during the examination process promoters of these sorts as part of the scope of their inventions.

How such broad claims could be granted so recently?

• Readers may wonder how patents with such broad claims directed to an enabling technology known and used for selection of transformed plants for at least more than a decade could have been granted at all. In this case, the answer is related to the earliest priority date claimed by the applicants for their inventions. The first filing related to the inventions was January 1983, a time when the development of vectors for transformation and methods to select the transformed cells was taking its first steps. Thus, although the applications for these particular patent documents were filed in 1995 and 1999 (see Bibliography table), the only published information that counts for assessing the novelty and non-obviousness of the inventions is that published before the earliest priority date claimed: that is, the relevant prior art to the inventions published prior to January 17, 1983. The myriad research and developments in this area published after January 17, 1983 may not be used by the patent office examiners in assessing patentability of the inventions.

Neomycin phosphotransferase (npt) gene

General aspects

Scientific info

Two neomycin phosphotransferase genes are used in selection of transformed organisms: the neomycin phosphotransferase I (nptI) gene and the neomycin phosphotransferase II (nptII) gene. The second one is the more widely used. It was initially isolated from the transposon Tn5 that was present in the bacterium strain Escherichia coli K12. The gene codes for the aminoglycoside 3'-phosphotransferase (denoted aph(3')-II or NPTII) enzyme, which inactivates by phosphorylation a range of aminoglycoside antibiotics such as:[add a comment]

• kanamycin
• neomycin
• geneticin (G418), and
• paromomycin.

NPTII is probably the most widely used selectable marker for plant transformation. It is also used in gene expression and regulation studies in different organisms in part because N-terminal fusions can be constructed that retain enzymatic activity. In animal cells, G418 and neomycin are used as selectable agents.[add a comment]

NPTII protein activity can be detected by enzymatic assay. In other detection methods, the modified substrates -the phosphorylated antibiotics- are detected by thin-layer chromatography, dot-blot analysis or polyacrylamide gel electrophoresis.[add a comment]

Plants such as maize, cotton, tobacco, Arabidopsis, flax, soybean and many others have been successfully transformed with the nptII gene. In plants, kanamycin is the most commonly used selective agent, normally in concentrations ranging from 50 to 500 mg/l. It is very effective in inhibiting the growth of untransformed cells. However, kanamycin is ineffective as a selection marker for several legumes and gramineae. For instance, in rice, kanamycin seems to interfere with the regeneration of transformed cells to green plants. As an alternative, paromomycin can be used for selecting nptII-transformed rice cells. Therefore, the choice of the selective agent is important and based on the plant species to be transformed.[add a comment]

Agricultural industrial applications

Field trials and commercial releases

According to information provided by BioTrack, a database administered by the Organisation for Economic Cooperation and Development (OECD) containing records of field trials and commercial releases in OECD countries (currently 30) from 1996 to 2000, essentially all of genetically-modified organisms (GMO) are plants (98.4%). Most of the research and development of GMOs is carried out in the United States (71.1%). The rest of the OECD countries contribution to GMOs is less than 10% each, with Canada close to 9% and the other countries ranging between 5% and 0.6%. Among plants, maize is the crop with the largest number of genetically-modified varieties (37.4%) followed by oilseed rape (12.4%) and potato (12.1%).[add a comment]

Most introduced traits in the modified crops confer resistance to compounds such as herbicides, pests, such as insects and nematodes, and diseases caused by bacteria, fungi and viruses. Characteristics such as color of flowers, delayed ripening of fruits, and sterility have also been introduced in plants to a lesser extent. Antibiotic resistance is not a trait of interest for most of the modified plants. Nevertheless, nptII is a feature present in many plant releases because it has been used to assist in their selection.[add a comment]

According to the information on globally approved GM plants compiled and provided by Agriculture & Biotechnology Strategies (Canada) Inc., modified plants containing nptII gene that are approved for release into the environment as food or feed products include maize, canola (oilseed rape), melon, potato, tomato and cotton as a fiber crop. Most of the releases have occurred in the United States. However, some transformed cotton varieties developed by Monsanto have been approved in several other countries such as Australia, Argentina, Canada, China, India, Japan, Mexico and South Africa.[add a comment]

Multiple risk assessments of crops, including those for human consumption, containing the nptII gene and its protein have found that there are no scientific reasons to deny or restrict the use of this gene in transgenic crops on grounds of human, animal or environmental safety.[add a comment]

IP aspects of the npt gene

Analysis of the filed and granted inventions

The selected analysed patents and patent applications related to the npt gene(s) are divided as follows:[add a comment]

• npt gene as part of a chimeric gene construct for plant transformation
• nptII gene in combination with paromomycin as a selective agent
  
• nptII gene as part of a bifunctional marker gene
• Aminoglycoside phosphotransferase gene conferring resistance to G418

npt gene as part of a chimeric gene construct for plant transformation.

Monsanto has two granted United States patents and one granted European patent claiming chimeric constructs for plant transformation containing a gene encoding a neomycin phosphotransferase enzyme, which confers antibiotic resistance to the transformed plant. The chimeric constructs of the inventions also comprise a promoter and a polyadenylation signal sequence. The United States patents 6174724 and 5034322 are also analysed in Antibiotic resistance genes in general.

One of the limitations of the inventions lies in the sort of promoter used in the chimeric construct to control the npt gene:

As discussed in the section Antibiotic resistance genes in general, a promoter "naturally expressed" in plants is not explicitly defined by the inventors. Yet the meaning of the term can be deduced from the description of the invention and the file history of the patent. Both sources tell us that the inventors may have envisioned any promoter from a gene of plant origin and promoters from genes of other organisms such as the nopaline synthase (nos) gene of A. tumefaciens, which is expressed only in a plant cell under natural conditions.

Opine genes are present in the Ti (tumor-inducing) plasmids and Ri (root-inducing) plasmids of Agrobacterium species. These genes are inactive while in the bacterial cells and are expressed only after they enter the plant cells. They code for enzymes that metabolize substances called "opines," such as octopine, nopaline, and agropine. Opines are utilized by the bacteria as a source of carbon, nitrogen, and energy. The promoter claimed in the US patent 5034322 can be derived from any of the opine genes present in the Ti plasmids of the species A. tumefaciens.

Ribulose-1,5-bis-phosphate carboxylase (Rbc) catalyzes the reduction of atmospheric CO2 during photosynthesis. In higher plants, Rbc is a protein composed of eight copies of chloroplast-encoded large subunits and eight copies of nuclear-encoded small subunits (ss). The promoter claimed in the United States patent 5034322 and the European patent is isolated from a gene encoding a small subunit. There is no limitation on the plant source of the gene; it can be derived from any plant.

The antibiotic resistance gene coding for neomycin phosphotransferase is not restricted to a particular gene sequence. The protected gene in the European patent codes for either a neomycin phosphotransferase I or neomycin phosphotransferase II. According to the invent ors, these are distinct enzymes with major differences in their amino acid sequences and substrate specificity. Thus, in the United States and in Europe, chimeric constructs designed for plant cells having any DNA sequence encoding a neomycin phosphotransferase could be encompassed by the claims.

View Independent Claims

npt gene as part of a chimeric gene construct for plant transformation

Patents granted to Monsanto

Actual granted independent claims

nptII gene in combination with paromomycin as a selective agent

Japan Tobacco has filed a European patent application directed to the use of the nptII gene in combination with the antibiotic paromomycin for the selection of transformed rice cells.

The nptII gene as a selectable marker for monocot plants, especially rice, has not been very effective when used in combination with kanamycin as a selective agent. This antibiotic negatively affects the regeneration of the transformed plants. The performance of G418 as a selective agent for monocots is better, but there is still poor transformation efficiency. The applicants claim that the combination of the nptII gene with paromomycin constitutes a highly efficient rice transformation system.

The filed claims of the European patent application are limited to:

• a method to select transformed rice cells, which contain a gene of interest and a paromomycin resistance gene by applying paromomycin to the medium;
• a method for producing rice transformants by Agrobacterium-mediated transformation of rice cells with a construct having a gene of interest and a paromomycin resistance gene. The transformed cells are selected with paromomycin and regenerated into complete plants; and
• transformed rice selected following the method mentioned above.

The paromomycin resistance gene is not limited to the nptII gene in the filed independent claims. Thus, the invention might encompass any gene that confers resistance to paromomycin, including the nptII gene. The use of paromomycin and a paromomycin resistance gene for the selection of rice plants is one of the main limitations of the invention as filed. It is not possible to ascertain the exact limitations of the claims as the application has not been granted yet.

View Independent Claims

nptII gene in combination with paromomycin as a selective agent

Patent filed by Japan Tobacco

Actual filed independent claims

nptII gene as part of a bifunctional marker gene

The National Research Council of Canada has two granted patents, in the United States and in Europe, directed to dual genetic markers composed of fused genes, which provide a reporter marker gene (glucuronidase (gusA) gene) and an antibiotic resistance gene (nptII gene).

The gusA gene encodes ß-glucuronidase (GUS), a hydrolase that cleaves a wide variety of ß-glucuronides. GUS is the most widely used reporter system for plants. It is easy to quantify, highly sensitive and very specific. Substrates for GUS are available for spectrometric, fluorometric and histochemical detection assays.

The bifunctional genetic marker of the invention allows for genetic selection of the transformed cells (npt II gene) and subsequent spatial localization and quantitative estimation of gene activity (gus gene).

The invention is limited with respect to the components of the fusion marker. However, the host organism expressing the marker is not limited to any organism in particular. It could potentially be any host as long as it is capable of expressing the stable polypeptide having both activities.

View Independent Claims

nptII gene as part of a bifunctional marker gene

Patents granted to the National Research Council of Canada

Actual granted independent claims

Aminoglycoside phosphotransferase gene conferring resistance to G418

There are several aminoglycoside phosphotransferases conferring resistance to aminoglycoside antibiotics. The aminoglycoside phosphotransferase I (aph-I) enzyme and the aminoglycoside (or neomycin) phosphotransferase II (APH-II or NPTII) are unrelated except for their ability to inactivate the antibiotic G418. The aph-I gene was originally found on transposon Tn601, also known as Tn903. According to some reports, aph-I is approximately four times more effective than aph-II in inactivating G418.

Cetus Corporation (acquired by Hoffman-La Roche in the early 90's) has two granted patents, one in the United States and one in Canada, on the DNA sequence of a modified aph-I enzyme. Modified truncated aph-I gene can be used as a selectable marker for both prokaryotic and eukaryotic organisms.

Both patents claim a truncated DNA sequence of an aph-I gene, which contains restriction sites immediately upstream of the start codon (Claim 1). This set-up ensures a precise reproducible translation of the gene and permits the construction of fusion proteins that contain the aph-I sequences at the C-terminal end. The aph-I gene of the invention is further modified by removing the codons for amino acids in positions 2-10, inclusive, and a couple of restriction sites within the codifying region. This modified truncated aph-I (mt aph -I) gene is particularly effective against G418. It also inactivates the antibiotic neomycin effectively, but it is less effective against kanamycin than the nptII (or aph-II) gene.

The Canadian patent further claims:

• a recombinant expression vector having the mt aph-I gene capable of conferring resistance to G418 on prokaryotes and eukaryotes (Claim 4);
• an expression vector with the mt aph-I gene as described under the control of promoter for eukaryotic cells (Claim 17);
• an expression vector for eukaryotes having a sequence for a fusion protein that has a mt aph-I gene as described in its C-terminal (Claim 19);
• a fusion protein that contains a C-terminal mt aph-I sequence and a N-terminal sequence of:
  • any desired protein (Claim 30);
  • ß-isopropylmalate dehydrogenase (Claim 31); and
  • yeast enolase (Claim 35).
• a method of purifying a fusion protein that comprises the mt aph-I gene as described (Claim 37).

There may be some marginal overlap between the United States patents filed by Monsanto and the United States patent granted to Cetus Corporation. Although Monsanto's patents are directed to chimeric genes, the DNA sequence coding for a neomycin phosphotransferase is not limited to any in particular. So, it might well include a neomycin phosphotransferase I (aph-I) gene or a neomycin phosphotransferase II (nptII or aph-II) gene.

The sequence claimed by Cetus Corporation has some limitations. It is a modified and truncated version of aph -I. Thus, it is likely that an aph-I gene without the modifications of the claimed version of the gene would not be encompassed by the claims.

View Independent Claims

Coding sequence for aminoglycoside phosphotransferase I (aph-I)

Patents granted to Cetus Corporation (U.S. patent) & Chiron Corporation (CA patent)

Actual granted independent claims

Hygromycin phosphotransferase (hpt) gene

General aspects

Scientific info

The hygromycin phosphotransferase (denoted hpt, hph or aphIV) gene was originally derived from Escherichia coli. The gene codes for hygromycin phosphotransferase (HPT), which detoxifies the aminocyclitol antibiotic hygromycin B. A large number of plants have been transformed with the hpt gene and hygromycin B has proved very effective in the selection of a wide range of plants, including monocotyledonous. Most plants exhibit higher sensitivity to hygromycin B than to kanamycin, for instance cereals. Likewise, the hpt gene is used widely in selection of transformed mammalian cells.[add a comment]

The sequence of the hpt gene has been modified for its use in plant transformation. Deletions and substitutions of amino acid residues close to the carboxy (C)-terminus of the enzyme have increased the level of resistance in certain plants, such as tobacco. At the same time, the hydrophilic C-terminus of the enzyme has been maintained and may be essential for the strong activity of HPT.[add a comment]

HPT activity can be checked using an enzymatic assay. A non-destructive callus induction test can be used to verify hygromycin resistance. The antibiotic hygromycin B should be handled with care because it is toxic to humans.[add a comment]

Agricultural industrial applications

Field trials and commercial releases[add a comment]

Like the nptII marker gene, hpt has been used as a selectable marker gene for transgenic plants, but it is not an agronomic trait of interest in modified plants. It is one of the introduced genes needed to accomplish the production of transformed plants.[add a comment]

According to information provided by BioTrack, a database administered by the Organisation for Economic Cooperation and Development (OECD) containing records of field trials and commercial releases in OECD Member countries (currently 30), several cultivars of rapeseed, alfalfa and canola submitted to field trials in the United States and Canada between 1989 and 1996 contained hygromycin phosphotransferase as a selectable marker gene.[add a comment]

In Australia, several varieties of barley, wheat, grapevine, Indian mustard and poppy, engineered for viral tolerance, fungal resistance, improvement of fruit quality and insect resistance contain a hygromycin phosphotransferase gene. These crops are being tested in field trials and have not been commercially released yet.[add a comment]

IP aspects of the hpt gene

Analysis of the protected inventions

The patents on the hpt gene and its applications in prokaryotic and eukaryotic transformation are a very interesting example of a comprehensive patent protection strategy followed by a company to consolidate an exclusive position in an enabling tool around the world.[add a comment]

Eli Lilly & Company has 22 granted patents (as of July 2002) in at least 10 different countries and a couple of patent applications that cover:[add a comment]

1. the wild-type, isolated hpt gene and vectors containing it for prokaryotic and eukaryotic expression;
2. a modified hpt gene; and
3. plasmids containing a modified hpt gene for plant transformation.

The patents granted in the United States, Canada and Germany have been assigned to Novartis (now Syngenta).[add a comment]

The patents are divided into three families according to their common priority applications and are directed to the following aspects:[add a comment]

• Family 1. Recombinant DNA cloning vectors having the hpt gene for eukaryotic and prokaryotic cell transformation
• Family 2. Modified hygromycin B resistance-conferring gene
• Family 3. Selectable marker for development of vectors and transformation systems in plants

Family 1. Recombinant DNA cloning vectors having the hpt gene for eukaryotic and prokaryotic cell transformation

Eli Lilly's portfolio of patents on this matter starts with this group, which discloses recombinant DNA cloning vectors that confer resistance to hygromycin (hyg B) and G418 antibiotics in both prokaryotic and eukaryotic cells. The patents encompass the initially isolated gene sequences for these antibiotic resistance genes.

The gene coding for an enzyme conferring resistance to G418 does not correspond to the nptI nor the nptII gene from the transposons Tn603 and Tn5, respectively. In this case it is a gene encoding an aminoglycoside acetyltransferase enzyme.

A cloning vector of the invention comprises:

1. a eukaryotic promoter;
2. structural gene(s) and its associated control sequence(s) for either hyg B or G418 resistance or both; and
3. a prokaryotic replicon.

When the host cell is a prokaryote, the antibiotic resistance genes and their control sequences are adjacent to the prokaryote replicon. In the case of a eukaryotic host cell, the eukaryotic promoter drives a single gene, either hpt or G418 resistance gene, but not both.

The plasmid pKC203 from the Escherichia coli JR225 strain is the parent plasmid harboring both antibiotic conferring-resistance genes used for the construction of a series of plasmids for use in transformation of prokaryotic and eukaryotic cells.

The plasmid pKC2O3 is the source of restriction fragments containing both or either one of the antibiotic resistance genes, which are then inserted into different plasmids. This series of plasmids are part of the claimed invention. The claimed fragments are:

• a 7.5 kb Bgl II fragment containing both resistance genes;
• a 2.5 kb Bgl II/Sal I fragment containing both resistance genes;
• a 1.51 kb Sac I/Bgl II fragment containing the hpt gene; and
• a 1.61 kb EcoR I/Sal I fragment containing the G418 resistance gene.

The antibiotic resistance genes of one of the recombinant cloning vectors are claimed in general terms without defining a specific DNA sequence (Claim 1 of the United States and Canadian patents, and Australian patent 555574 B). In these countries, the invention is likely to cover any DNA sequence encoding the enzymes against these antibiotics.

Transformed prokaryotic and eukaryotic host cells are also the subject of independent claims. Eukaryotic host cells include mouse, Escherichia coli, Saccharomyces cerevisiae and human. It does not mean, however, that the recombinant vectors of the invention are only limited to this group of hosts. Other independent claims encompass eukaryotes and prokaryotes in general. Thus, virtually any organism could be covered by the invention.

All the above mentioned features are the subject matter of the Australian, Canadian, European and United States patents. In addition, the United States patent claims the amino acid sequence of the hygromycin phosphotransferase (HPT) enzyme. Patents granted in other countries were not analysed.

View Independent Claims

Patent Family No. 1
Recombinant DNA cloning vectors having the hpt gene for eukaryotic and prokaryotic cell transformation

Patents granted to Eli Lilly Co.

Actual granted independent claims

This patent was assigned to Novartis