Title: Arabitol or ribitol as positive selectable markers
Abstract: Disclosed herein are novel methods and materials for selecting transgenic cells. Specifically exemplified herein are positive selection methods that involve conferring to cells the ability to metabolize certain compounds, preferably arabitol, ribitol, raffinose, sucrose, mannitol or combinations thereof. Accordingly, transformed cells can be selected by simply subjecting them to a medium containing such compounds. The subject invention alleviates the disadvantages and concerns of negative selection methods, such as the unnecessary killing of transformed cells and the dispersal of potentially harmful genes (e.g., antibiotic or herbicide resistant genes) into the environment. Furthermore, novel nucleotide sequences relating to the E. coli rtl operon and arabitol dehydrogenase gene, and amino acid sequences relating to the gene products thereof are disclosed.
Patent Number: 7,005,561 Issued on 02/28/2006 to Parrott, et al.
| Inventors:
|
Parrott; Wayne (Athens, GA);
LaFayette; Peter (Watkinsville, GA);
Kane; Patrick (Athens, GA)
|
| Assignee:
|
University of Georgia Research Foundation, Inc. (Athens, GA)
|
| Appl. No.:
|
802208 |
| Filed:
|
March 8, 2001 |
| Current U.S. Class: |
800/288; 435/419; 435/468; 435/471; 435/483; 435/484; 536/23.7; 800/298 |
| Current Intern'l Class: |
C12N 15/82 (20060101); C12N 15/74 (20060101); C12N 15/90 (20060101); A01H 5/10 (20060101) |
| Field of Search: |
435/3201,410,419,468,471,483,484
536/237
800/278,288,295,298
|
References Cited [Referenced By]
U.S. Patent Documents
| 4857467 | Aug., 1989 | Sreekrishna et al.
| |
| 5011909 | Apr., 1991 | Borovsky et al.
| |
| 5130253 | Jul., 1992 | Borovsky et al.
| |
| 5223419 | Jun., 1993 | Katagiri et al.
| |
| 5254801 | Oct., 1993 | Dotson et al.
| |
| 5767378 | Jun., 1998 | Bojsen et al.
| |
| Foreign Patent Documents |
| WO 942627 | Sep., 1994 | WO.
| |
| WO 974448 | Nov., 1997 | WO.
| |
Other References
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|
Primary Examiner: Mehta; Ashwin
Attorney, Agent or Firm: Van Dyke; Timothy H., Wolter; Beusse Brownlee, Mora & Maire
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 USC §119(e) of Provisional
Application Nos. 60/188,291 filed Mar. 8, 2000 and 60/225,595 filed Aug. 15, 2000.
Claims
What is claimed is:
1. An isolated polynucleotide molecule comprising at least one gene of interest,
and at least one selectable marker gene, wherein said at least one selectable marker
gene comprises a nucleotide sequence which selectively hybridizes under high stringency
conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 2, or
a plant optimized version thereof, wherein said nucleotide sequence encodes for
a protein possessing ribitol dehydrogenase enzymatic activity and a protein possessing
ribitol kinase enzymatic activity.
2. The Transgenic cells transformed with the polynucleotide molecule of claim
1, wherein the selectable marker gene gives said cells a selective advantage when
a population of cells including the transformed cells and nontransformed cells
is supplied with a marker compound.
3. The transgenic cells of claim 2 wherein said marker compound is arabitol,
ribitol, or mannitol.
4. The transgenic cells of claim 2, wherein said transgenic cells comprise bacteria,
fungi, yeast, plant or a combination thereof, and wherein said nucleotide sequence
is optimized for expression in said cells.
5. A Plant or plant tissue regenerated from the cells of claim 2.
6. A method of selecting transformed cells from a population of cells comprising
a) introducing into the genome of a cell a gene of interest and a selectable
marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed
cells have a selective advantage over non-transformed cells due to expression or
transcription of the the selectable marker gene in the presence of the marker compound;
and
d) selecting said transformed cells from the population of cells;
wherein said selectable marker gene comprises a nucleotide sequence which selectively
hybridizes under high stringency conditions to the complement of a nucleotide sequence
shown in SEQ ID NO: 2, or a plant optimized version thereof, wherein said nucleotide
sequence encodes a protein that possesses ribitol dehydrogenase enzymatic activity
and a protein that possesses ribitol kinase enzymatic activity;
and said marker compound comprises arabitol, ribitol, or mannitol.
7. The method of claim 6, wherein said cells comprise bacteria, fungi, yeast,
plant or a combination thereof, and wherein said nucleotide sequence optimized
for expression in said cells.
8. The method of claim 7, wherein said cells comprise plant cells.
9. Transformed cells selected according to the method of claim 6.
10. Transformed plants derived from the cells of claim 9.
11. Seeds produced from the transformed plants of claim 10, wherein said seeds
are capable of germinating to produce transformed plants.
12. An isolated polynucleotide molecule comprising a nucleotide sequence which
selectively hybridizes under high stringency conditions to the complement of a
plant optimized version of the nucleotide sequences shown in SEQ ID NO: 2, and
wherein said nucleotide sequence encodes for a protein possessing ribitol dehydrogenase
activity and a protein possessing ribitol kinase activity.
13. An isolated polynucleotide molecule comprising at least one gene of interest,
and at least one selectable marker gene, wherein said at least one selectable marker
gene comprises a nucleotide sequence encoding SEQ ID NOS.: 3 and 4.
14. An isolated polynucleotide molecule comprising at least one gene of interest,
and at least one selectable marker gene, wherein said at least one selectable marker
gene comprises a nucleotide sequence which selectively hybridizes under high stringency
conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 1, or
a plant optimized version thereof, wherein said at least one selectable marker
gene encodes for a protein possessing arabitol dehydrogenase enzymatic activity.
15. A method of selecting transformed cells from a population of cells comprising
a) introducing into the genome of a cell a gene of interest and a selectable
marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed
cells have a selective advantage over non-transformed cells due to expression or
transcription of the selectable marker gene in the presence of the marker compound;
and
d) selecting said transformed cells from the population of cells;
wherein said selectable marker gene comprises a nucleotide sequence which selectively
hybridizes under high stringency conditions to the complement of a nucleotide sequence
shown in SEQ ID NO: 1, or a plant optimized version thereof, and encodes a protein
having arabitol dehydrogenase enzymatic activity;
and wherein said marker compound is arabitol.
16. A method of selecting transformed cells from a population of cells comprising
a) introducing into the genome of a cell a gene of interest and a selectable
marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed
cells have a selective advantage over non-transformed cells due to expression or
transcription of the selectable marker gene in the presence of the marker compound;
and
d) selecting said transformed cells from the population of cells;
wherein said selectable marker gene comprises a nucleotide sequence encoding
SEQ ID NO.: 3, and a nucleotide sequence encoding SEQ ID NO.: 4;
and wherein said marker compound is ribitol.
17. The method of claim 16, wherein said selectable marker gene further comprises
a nucleotide sequence encoding SEQ ID NO.: 5.
18. The isolated polynucleotide molecule of claim 1, wherein said nucleotide
sequence further encodes a protein possessing ribitol transporter activity.
19. The isolated polynucleotide molecule of claim 1, wherein codons of the nucleotide
sequence that hybridizes to the complement of the nucleotide sequence of SEQ ID
NO: 2 are substituted with plant preferred codons.
20. The method of claim 6, wherein codons of the nucleotide sequence that hybridizes
to the complement of the nucleotide sequence of SEQ ID NO: 2 are substituted with
plant preferred codons.
21. The isolated polynucleotide molecule of claim 12, wherein codons of the nucleotide
sequence that hybridizes to the complement of the nucleotide sequence of SEQ ID
NO: 2 are substituted with plant preferred codons.
22. The isolated polynucleotide molecule of claim 14, wherein codons of the nucleotide
sequence that hybridizes to the complement of the nucleotide sequence of SEQ ID
NO: 1 are substituted with plant preferred codons.
23. The method of claim 15, wherein codons of the nucleotide sequence that hybridizes
to the complement of the nucleotide sequence of SEQ ID NO: 1 are substituted with
plant preferred codons.
Description
BACKGROUND OF THE INVENTION
The term "transformation" is generally understood in the biotech and chemical
arts to refer to a stable incorporation of a foreign DNA or RNA into a cell which
results in a permanent, heritable alteration in the cell. It is well known that
when new genetic material is to be introduced into a population of cells by transformation,
only a certain number of the cells are successfully transformed. It is then necessary
to identify the genetically transformed cells so that these cells can be separated
from the non-transformed cells of the population. Identification and separation
of the transformed cells has traditionally been accomplished using "negative selection",
whereby the transformed cells are able to survive and grow, while the non-transformed
cells are subjected to growth inhibition or perhaps even killed by a substance
which the transformed cells, by virtue of their transformation, are able to tolerate.
For example, when a population of plant cells is transformed, selection of the
transformed cells typically takes place using a selection gene which codes for
antibiotic or herbicide resistance. The selection gene—which in itself generally
has no useful function in the transformed plant (and may in fact be undesirable
in the plant) is coupled to or co-introduced with the desired gene to be incorporated
into the plant, so that both genes are incorporated into the population of cells,
or rather into certain of the cells in the population, since it is difficult, if
not impossible, in practice to transform all of the cells. The cells are then cultivated
on or in a medium containing the antibiotic or herbicide to which the genetically
transformed cells are resistant by virtue of the selection gene, thereby allowing
the transformed cells to be identified, since the non-transformed cells—which
do not contain the antibiotic or herbicide resistance gene in question—are
subjected to growth inhibition or are killed.
These negative selection methods have, however, certain disadvantages. First
of all, the non-transformed cells may die because of the presence of antibiotics
or herbicides in the growth medium. As a result, when the population of cells is
a coherent tissue there is a risk that not only the non-transformed cells but also
the transformed cells may die, due to the fact that the death of the non-transformed
cells may cut off the supply of nutrients to the transformed cells or because the
damaged or dying non-transformed cells may excrete toxic compounds.
Another disadvantage of negative selection is that the presence of an unnecessary
gene, for example antibiotic resistance, may be undesirable. There is concern among
environmental groups and governmental authorities about whether it is safe to incorporate
genes coding for antibiotic resistance into plants and microorganisms. This concern
is of particular significance for food plants and for microorganisms which are
not designed to be used in a closed environment (e.g. microorganisms for use in
agriculture), as well as for microorganisms which are designed for use in a closed
environment, but which may accidentally be released therefrom.
Positive selection is a selection system whose operating principle is converse
to negative selection. Rather than conferring resistance to a negative or toxic
substance, positive selection involves conferring onto the transformed cell a metabolic,
or other, competitive advantage over nontransformed cells. Positive selection systems
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. As alluded to above, there is increasing concern
that genes conferring resistance to antibiotics and/or herbicides may disperse
and be incorporated into agriculturally destructive weeds and other plants, as
well as pathogenic bacteria. Indeed, transgenic plants have been banned in the
European Union. As a result, more and more investigative efforts are being made
to develop positive selection systems for use in plants and other cell types.
SUMMARY OF THE INVENTION
The subject invention relates to a positive selection system that involves conferring
to transferred cells the ability to metabolize arabitol, ribitol, and/or mannitol.
One aspect of the subject invention pertains to a gene construct comprising a gene
of interest and a selectable marker gene. A specific aspect pertains to supplying
to a population of cells at least one marker compound which is directly or indirectly
active in the transformed cells containing the gene of interest and is inactive
or less active in the non-transformed cells whereby the transformed cells are provided
with a selective advantage. Alternatively, the selective advantage is one wherein
the expression of the gene of interest or the positive selecting gene leads to
an increase in the activity of an enzyme found endogenously in the population of
cells such that the activity of the enzyme in the transformed cells is greater
than the activity of the enzyme in non-transformed cells.
According to a further aspect, the marker compound supplied to the population
of cells is selected from the group consisting of arabitol, ribitol, mannitol or
a derivative or variant thereof.
A further aspect pertains to transformed cells selected according to the above-recited
method. Moreover, an additional aspect pertains to plants derived from said transformed cells.
According to an alternative aspect, the invention includes both positive
selection and negative selection including the use of a gene coding for antibiotic
or herbicide resistance.
Yet a further aspect of the invention relates to genetically transformed cells
comprising a gene of interest and selectable marker gene wherein the selectable
marker gene induces a positive effect in the transformed cells and gives said cells
a selective advantage when a population of cells including the transformed cells
and nontransformed cells is supplied with a compound.
Moreoever, another aspect of the invention includes a method of selecting
genetically transformed cells from a population of cells comprising
a) introducing into the genome of a cell a gene of interest and a selectable
marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed
cells have a selective advantage over non-transformed cells due to expression or
transcription of the gene of interest or the selectable marker gene in the presence
of the marker compound; and
d) selecting said transformed cells from the population of cells wherein said
selectable marker gene comprises a ribitol or D-arabitol dehydrogenase, a ribitol
or D-arabitol kinase, a ribitol or D-arabitol transporter gene, or a combination
thereof, and the compound is arabitol, ribitol or a derivative precursor thereof.
Further still, an additional aspect of the subject invention pertains to
polynucleotide molecules that encode proteins having the biological activity of
ribitol or arabitol dehydrogenase, ribitol or arabitol kinase, ribitol or arabitol
transporter, or ribitol or arabitol repressor. Specifically, the aspect pertains
to a polynucleotide as shown in SEQ ID NOS: 1 and 2, or functional fragments and
variants thereof. Furthermore, another aspect of the subject invention pertains
to a polypeptide encoded by the polynucleotide molecules of the subject invention.
A further aspect of the subject invention pertains to cells transformed with
the
polynucleotide molecules of the subject invention. Specifically exemplified are
transformed bacteria, fungi, yeast, animal and plant cells. More specifically exemplified
are transformed bacteria and plant cells.
These and other advantageous aspects of the subject invention are described
in further detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic of the isolation and cloning of the
E. coli rtl operon.
FIG. 2 shows a graph representing comparative plasmid yields in DH10B of pBluescript
and pMECA, growing in LB broth or 2B broth minimal medium supplemented with glucose,
and of their ribitol derivatives growing on 2B minimal medium with ribitol. Plasmid
yield data were collected at 17 and 41 hours. Plasmid yields for pBluescript-R
and pMECA-R with a GUS construct cloned into their multiple cloning sites were
also compared. Bars represent the average of three replications +/- standard error.
Legend: The first letter signifies the plasmid backbone: B=pBluescript, M=pMECA.
The second letter indicates the medium:L=Luria-Bertani broth, M=2B minimal medium.
The third position denotes the carbohydrate source: a '-' means no additional carbohydrate,
G=glucose, and R=ribitol. The number is for the amount of growth time: 17 or 41
hours. The final position denotes if the plasmid had an insert cloned into its
multiple cloning site: a '-' means no insert, and G refers to a GUS construct.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 represents the nucleotide sequence of the arabitol dehydrogenase
gene from
E. coli strain C.
SEQ ID NO: 2 represents a polynucleotide molecule which comprises a nucleotide
sequence encoding the rtl operon from
E. coli strain C. Bases 96 to 848
encode for ribitol dehydrogenase. Bases 859 to 2463 encode for ribitol kinase.
Bases 2565 to 3839 encode for ribitol transporter.
SEQ ID NO: 3 represents the amino acid sequence of
E. coli strain C ribitol dehydrogenase.
SEQ ID NO: 4 represents the amino acid sequence of
E. coli strain C ribitol kinase.
SEQ ID NO: 5 represents the amino acid sequence of
E. coli strain C ribitol transporter.
DETAILED DISCLOSURE OF THE INVENTION
The term "gene of interest" as used herein refers to any nucleotide sequence,
which is to be incorporated into the cells to produce genetically transformed cells.
Introduction of nucleotide sequence into plants, microorganisms and animals is
widely practiced, and there are no limitations upon the nucleotide sequences whose
presence may be detected by use of the positive selection method described herein.
By use of the subject methods the presence of the gene of interest in the genetically
transformed cells may be determined without the above-mentioned disadvantages associated
with traditional negative selection systems.
The term "selectable marker gene" refers to any nucleotide sequence that is preferably
co-introduced with a gene of interest, wherein a selective advantage is conferred
to a cell transformed with said selectable marker gene.
In a preferred embodiment, the gene of interest is directed to one or more functional
genes that are chosen to provide a new plant trait, to enhance an existing plant
trait, or to otherwise modify expression of plant phenotypes exhibited by the plant.
Such traits include herbicide resistance, pesticide resistance, disease resistance,
environmental tolerance (e.g., heat, cold, drought, salinity), morphology, growth
characteristics, nutritional content, taste yield, horticultural characteristics,
consumer (quality) traits, and the like.
A functional gene to be introduced may be a structural gene which encodes a polypeptide
which imparts the desired phenotype. Alternatively, the functional gene may be
a regulatory gene which might play a role in transcriptional and/or translational
control to suppress, enhance, or otherwise modify the transcription and/or expression
of an endogenuous gene within the plant. It will be appreciated that control of
gene expression can have a direct impact on the observable plant characteristics.
Often the functional genes to be introduced will be modified from their native
form. For example, sense and anti-sense constructs referred to above often have
all or a portion of the transcript of the native gene operably linked to a promoter
sequence at the 5′ end of the transcribable segment, and operably linked
to the 3" sequence of another gene (including polyadenylation sequences) at the
3′ end of the transcribable segment. As is apparent to those skilled in
the art, the promoter sequence could be one of the many plant active sequences
already described. Alternatively, other plant-active promoter sequences could be
derived specifically to be linked to the transcribable segment. The promoter can
be endogenous to a particular plant species, or can be from an exogenous source
such as a cauliflower mosaic virus 35S promoter (Odell et al., Nature 313:810-812
(1985)), the ubiquitin 1 promoter, or the Smas promoter. The 3′ end sequence
to be added can be derived from another plant gene, or less preferably from any
other eukaryotic gene.
The fact that a gene of interest is co-introduced with a selectable marker gene
refers to the fact that the sequences are coupled to each other or otherwise introduced
together in such a manner that the presence of the selectable marker gene in a
cell indicates that the gene of interest has been introduced into the cell. The
two nucleotide sequences are typically, although not necessarily, part of the same
genetic construct and are introduced by the same vector. A genetic construct containing
the two nucleotides sequences will typically, but not necessarily, contain regulatory
sequences enabling expression of each nucleotide sequence for example, promoter
and transcription terminators.
The term "cells" within the context of the present invention is intended to refer
to any type of cells from which individual genetically transformed cells may be
identified and isolated using the method of the invention, and includes cells of
plants, animals and microorganisms such as bacteria, fungi, yeast, etc. Furthermore,
the term cell is includes protoplasts. Particularly preferred cells are plant cells
and bacteria. More particularly the transformed plant cells and plants, seeds or
progeny derived therefrom include: fruits such as tomato, mango, peach, apple,
pear, strawberry, banana and melon; field crops such as canola, sunflower, tobacco,
soybean and sugar beet; small grain cereals such as wheat, barley, rice, corn,
and cotton; ornamentals; forages such as alfalfa, clover, forage grasses; forest
trees; and vegetables crops such as potato, carrot, lettuce, cabbage and onion.
Most preferably are soybean and corn.
The term "population of cells" refers to any group of cells which has be subjected
to genetic transformation. The population may be a tissue, an organ or a portion
thereof, a population of individual cells in or on a substrate, for example, a
culture of microorganism cells, or a whole organism, for example, an entire plant.
The term "selecting" refers to the process of identifying and/or isolating genetically
transformed cells from the non-transformed cells in a population of cells using
the methods disclosed herein.
The gene of interest and the selectable marker gene may be introduced independently.
The same bacteria may be used for incorporation of both genes and incorporating
a relatively large number of copies of the gene of interest into the cells, whereby
the probability is relatively high that cells which are shown to express the selectable
marker gene also will contain and express the gene of interest. Independent introduction
of two or more genes resulting in co-expression of the genes in the same cell is
generally expected to have low probability, and the improved selection frequencies
obtained by the positive selection method described herein are therefore expected
to be especially advantageous in such systems.
The term "marker compound" as used herein may be any compound or nutrient in
inactive or precursor form which in the absence of, for example, expression of
the selectable marker gene exists in a form which is substantially biologically
inactive with respect to the cells in question, but which when the selectable marker
gene is expressed or transcribed is hydrolyzed or otherwise activated or metabolized
so as to provide the genetically transformed cells containing the gene of interest
with a selective advantage, and thereby allowing the cells to be selected. Preferred
compounds include, but are not limited to, arabitol or ribitol and derivatives
or precursors thereof, and alternatively mannitol and derivatives and precursors
thereof. A "derivative" of arabitol or ribitol refers to any compounds capable
of being utilized by, binding to, being a substrate for, or a product of any protein
involved, either directly or indirectly, in the metabolism of arabitol or ribitol.
The marker compound used in the invention need not be one which is activated
directly by a polypeptide encoded by the selectable marker gene. It may be activated
indirectly, for example whereby the selectable marker gene has an indirect effect
upon the marker compound in genetically transformed cells but not in non-transformed
cells. Thus, the selectable marker gene may be one which upon expression in the
transformed cells, for example, indirectly increases the activity of an enzyme
which is endogenous to the population of cells, thereby leading to a greater enzyme
activity and activation of the compound in question in the genetically transformed cells.
The term "selective advantage" as used herein includes the terms selective, metabolic
and physiological advantage and means that the transformed cells are able to grow
more quickly than disadvantaged (non-transformed) cells, or are advantageously
able to utilize substrates (such as nutrient precursors, etc.) which disadvantaged
cells are not able to utilize, or are able to detoxify substrates which are toxic
or otherwise growth inhibitory to disadvantaged cells or a combination thereof.
However, the non-transformed cells do not necessarily suffer any severe disadvantage
in the sense of being damaged or killed or as is the case with negative selection
using antibiotics or herbicides.
Therefore the positive selection as used in the context of the present
invention refers to the use of a selectable marker gene which produces or increases
a positive effect of an added compound on the transformed cells.
A protein which is "involved in the metabolism of a marker compound" is typically,
but not exclusively, an enzyme which may be responsible directly or indirectly
for the production or utilization of the marker compound or its derivatives or
precursors. The protein may also be involved in the metabolism of a marker compound
if it binds to it, transfers it from one site to another within or transport into
the cell or tissue or organism or otherwise sequesters it thereby altering its
local availability.
A region of nucleotide sequence which "regulates the activity of a gene encoding
a protein" may alter the level of expression of an endogenous gene by being a promoter,
or having a promoter activity therefor, and by being introduced in or near its
vicinity. By "near" is meant up to 10,000 kb. Alternatively, indirect regulation
may arise by altering the binding of RNA polymerase to the promoter of a structural
gene encoding a protein, or complementary binding of the nucleotide sequence to
at least a part of the structural gene, thus typically reducing the quantity of
the protein in the cell.
Use of the present positive selection method in vivo is of particular relevance,
for example, in connection with transformation performed on whole plants or on
plant parts, in which the plants or parts comprise both transformed and non-transformed
cells, since selection of the transformed cells is achieved without directly damaging
the neighboring non-transformed cells. The transformed cells thus have a selective
"advantage" compared to the non-transformed cells (e.g. the ability to thrive and
grow; in plants, e.g., the ability to form shoots, etc.), but the non-transformed
cells do not suffer any severe disadvantage in the sense of being damaged or killed,
as in the case with negative selection using antibiotics or herbicides.
The selective advantage possessed by the transformed cells may typically be a
difference or advantage allowing the transformed cells to be identified by simple
visual means, i.e. without the use of a separate assay to determine the presence
of a marker gene.
A population of cells may be cultivated on or in a medium containing at least
one
compound which may be inactive and which is directly or indirectly activated in
the transformed cells, the compound being inactive in non-transformed cells or
less active in non-transformed cells than in transformed cells, such that the transformed
cells are provided with a selective advantage allowing them to be selected from
the cell population.
The population of cells may also be cultivated on or in a medium containing a
compound which is made available for the transformed cells by expression or transcription
of the nucleotide sequence, the compound not being available for the non-transformed
cells or being less available for non-transformed cells, such that the transformed
cells are provided with a selective advantage.
The cells may also be transformed with a selectable marker gene which may encode
a permease or other transport factor which allows the marker compound to cross
the cell membrane and enter the transformed cells or to cross another (organelle)
membrane, so that "activation" of an inactive compound involves selective uptake
of the compound by transformed cells, and uptake by non-transformed cells is not
possible or takes place to a lesser extent. Instead of facilitating uptake of a
compound into the cell, the positive selection gene may alternatively direct its
product to a compartment in which the inactive compound is located, for example,
outside the plasma membrane or into the vacuole or the endoplasmic reticulum.
A compound used for selection purposes may in addition have both a positive and
a negative effect. For example, certain carbon sources in sufficiently high concentrations
can be toxic to most plants, but in cells containing arabitol or ribitol metabolizing
enzymes, the negative effect is eliminated and the cells further obtain the benefit
of being able to use these compounds as a carbohydrate source. In this case a single
compound and a single or group genes together provide a combined positive and negative
selection system, although such a system may also be established using two or more
genes which together are responsible for inhibition of the negative effects of
a compound and manifestation of the positive effects of the compound in the transformed cells.
The cells may be transformed with any nucleotide sequence which it is desired
to incorporate therein to. Such a nucleotide sequence may encode genes providing
for viral, fungal, bacterial or nematode resistance.
The protein encoded by the gene of interest or preferably the selectable marker
gene is preferably an enzyme involved in arabitol or ribitol metabolism. Such enzymes
include ribitol or D-arabitol dehydrogenease, ribitol or D-arabitol kinase, or
D-ribitol or D-arabitol transporter gene. Scangos and Reiner,
Journal of Bacteriology,
134:492-500 (1978).
Examples of compounds which can exert a physiological effect upon entering
the cell, but which are not easily taken up into the cell or a cell compartment,
are strongly hydrophilic or hydrophobic compounds, in particular charged compounds,
large molecules such as polymers, in particular proteins, peptides, oligo- and
polysaccharides, including plant hormones, phosphorylated metabolites such as phosphorylated
carbohydrates, phosphorylated vitamins, phosphorylated nucleosides, including cytokinins,
and compounds which are conjugated to carboxylic acid-containing carbohydrates
or amino acids, including plant hormone conjugates.
Also, it is contemplated that the basic method of the present invention may
be modified so that, instead of activating an inactive compound or nutrient in
the transformed cells, selection may be performed by blocking the metabolism or
synthesis of a compound in these cells.
When a polypeptide encoded by the selectable marker gene or the gene of interest
directly activates an inactive compound or nutrient in the transformed cells, the
non-transformed cells may in certain cases contain or produce a certain amount
of the polypeptide in question. For example, when the activating polypeptide is
an enzyme, the non-transformed cells may contain a certain native enzyme activity,
the native enzyme being of the same type as the introduced activating enzyme. In
such cases the "inactive compound or nutrient" need not necessarily be completely
inactive in the non-transformed cells, since it may be sufficient that the compound
or nutrient is merely substantially less active in non-transformed cells than in
transformed cells. In other words, a qualitative difference between the transformed
cells and the non-transformed cells with regard to activation of the initially
inactive compound or nutrient may in certain cases be sufficient for selection
purposes. In such cases inhibitors or substrates which compete with the native
enzymes may be added. Especially suitable are inhibitors activated by the native
enzyme, resulting in self-catalyzed production of the active inhibitor to a level
at which the native enzyme is substantially totally inhibited.
The various methods employed in the preparation of the plasmids and transformation
of host organisms are well known in the art and are described, for example, in
U.S. Pat. Nos. 5,011,909 and 5,130,253. These patents are incorporated herein by
reference. These procedures are also described in Sambrook et al.,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989). Methods of transformation for use in accord with the subject
invention can include those conventional to the art, such as use of Agrobacterium,
viral vectors, microinjection, PEG, biolistics, and electroporation which are all
routinely used to introduce foreign DNA into plant cells. The mode of transformation
is not necessarily critical to the subject methods. Those skilled in the art will
appreciate that as other transformation methods are developed, these new transformation
methods can be practiced in accord with the teachings herein. Once in the cell,
the foreign DNA is incorporated into the plant genome. In a particular embodiment,
the transformation contemplates constructing a vector comprising a gene of interest
and a selectable marker gene, placing the vector into a selected strain of Agrobacterium,
and treating selected plant cells with the
Agrobacterium under conditions
sufficient to result in transfer of at least some of the vectors from the Agrobacterium
to the plant cells, whereby the polynucleotide is expressed in the plant cells.
Regulatory sequences can include both promoter and termination sequences.
Possible regulatory sequences can include, but are not limited to, any promoter
already shown to be constitutive for expression, such as those of viral origin
(CaMV 19S and 35S, TMV, AMV) or so-called "housekeeping" genes (ubiquitin, actin,
tubulin) with their corresponding termination/poly A+sequences. Also, seed-and/or
developmentally-specific promoters, such as those from plant fatty acid/lipid biosynthesis
genes (ACPs, acyltransferases, desaturases, lipid transfer protein genes) or from
storage protein genes (zein, napin, cruciferin, conglycinin, or lectin genes, for
example), with their corresponding termination/poly A+sequences can be used for
targeted expression. In addition, the gene can be placed under the regulation of
inducible promoters and their termination sequences so that gene expression is
induced by light (rbcS-3A, cab-1), heat (hsp gene promoters) or wounding (mannopine,
HGPGs). It is clear to one skilled in the art that a promoter may be used either
in native or truncated form, and may be paired with its own or a heterologous termination/polyA+sequence.
Plant tissue for use in transformation may be obtained from any suitable plant,
i.e., known to be susceptible to transformation by known methods. Appropriate plant
tissue includes, but is not limited to, leaves, hypocotyls, cotyledons, stems,
callus, single cells, and protoplasts.
In a particular embodiment, transformed callus tissue is selected by growth on
selection medium (e.g., medium which contains carbon source only utilizable by
transformed plant cells). Transformed plants are regenerated and screened for the
presence of the gene of interest. This involves analyzing tissue by at least one
molecular or biological assays to determine which, if any, transformants contained
the gene of interest. These assays include assays or observation of the tissue
for growth, and assays of the tissue for the presence of gene of interest by, for
example, a Southern assay or a PCR assay.
Those plants which are positive for the gene of interest are grown to maturity,
and tissue can be analyzed for the expression of the gene of interest by looking
for the polypeptide encoded by the polynucleotide, as for example via a Western
blot analysis, and for the phenotype conferred to the plant by the gene of interest.
It is now well known in the art that when synthesizing a gene for improved expression
in a host cell it is desirable to design the gene such that its frequency of codon
usage approaches the frequency of preferred codon usage of the host cell. For purposes
of the subject invention, "frequency of preferred codon usage" refers to the preference
exhibited by a specific host cell in usage of nucleotide codons to specify a given
amino acid. To determine the frequency of usage of a particular codon in a gene,
the number of occurrences of that codon in the gene is divided by the total number
of occurrences of all codons specifying the same amino acid in the gene. Similarly,
the frequency of preferred codon usage exhibited by a plant cell can be calculated
by averaging frequency of preferred codon usage in a large number of genes expressed
by the plant cell. It is preferable that this analysis be limited to genes that
are highly expressed by the host cell.
Thus, in one embodiment of the subject invention, plant cells can be genetically
engineered, e.g., transformed with genetic contructs to attain desired expression
levels of the gene of interest. To provide genes having enhanced expression, the
DNA sequence of the gene of interest can be modified to comprise codons preferred
by highly expressed genes to attain an A+T content in nucleotide base composition
which is substantially that found in the transformed host cell. It is also preferable
to form an initiation sequence optimal for said plant cell, and to eliminate sequences
that cause destabilization, inappropriate polyadenylation, degradation and termination
of RNA and to avoid sequences that constitute secondary structure hairpins and
RNA splice sites. For example, in synthetic genes, the codons used to specify a
given amino acid can be selected with regard to the distribution frequency of codon
usage employed in highly expressed genes in the plant cell to specify that amino
acid. As is appreciated by those skilled in the art, the distribution frequency
of codon usage utilized in the synthetic gene is a determinant of the level of expression.
In a preferred embodiment, the selectable marker genes pertain to SEQ ID NO.:
1 and SEQ ID NO.:2 as well as fragments or functional mutants thereof that are
capable of metabolizing a marker compound to confer a selective advantage. Such
fragments and mutants will be readily obtainable following the teachings herein
coupled with the state of the art. For example, using specifically exemplified
polynucleotides as probes, useful polynucleotides can be obtained under conditions
of appropriate stringency. The present invention further relates to variants of
the present polynucleotides which encode for fragments, analogs and derivatives
of the polypeptides having the sequences shown in SEQ ID NO.: 3, SEQ ID NO.:4,
and SEQ ID NO.:5. A variant of the polynucleotide may be a naturally occurring
variant such as a naturally occurring allelic variant, or it may be a variant that
is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide
may be made by mutagenesis techniques, including those applied topolynucleotides,
cells or organisms.
Among variants in this regard are variants that differ from the aforementioned
polynucleotides by nucleotide substitutions, deletions or additions. The substitutions
may involve one or more nucleotides. The variants may be altered in coding or non-coding
regions or both. Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Among the particularly preferred embodiments of the invention in this regard
are polynucleotides encoding polypeptides having the amino acid sequences shown
in SEQ ID NO.: 3, SEQ ID NO.:4, and SEQ ID NO.:5; variants, analogs, derivatives
and fragments thereof.
Further particularly preferred in this regard are polynucleotides encoding
one or more gene products of the ribitol or arabitol operons (e.g., RtlT, RtlK,
RtlD, RtlR, AtlD proteins), or combinations thereof, and fragments, and variants,
analogs and derivatives of the fragments, which have the amino acid sequences exemplified
herein in which several, a few, 1 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid
residues are substituted, deleted or added, in any combination. Especially preferred
among these are silent substitutions, additions and deletions, which do not alter
the properties and activities of the proteins. Also especially preferred in this
regard are conservative substitutions. Most highly preferred are polynucleotides
encoding polypeptides shown in SEQ ID NO.: 3, SEQ ID NO.:4, and/or SEQ ID NO.:5
without substitutions.
Further preferred embodiments of the invention are polynucleotides that are
greater than 79%, preferably at least 85%, more preferably at least 90% identical
to a polynucleotide encoding SEQ ID NO.: 1 and SEQ ID NO.:2, and polynucleotides
which are complementary to such polynucleotides. Among these particularly preferred
polynucleotides, those with at least 90%, 95%, 98% or at least 99% identity are
especially preferred.
Particularly preferred embodiments in this respect, moreover, are polynucleotides
which encode polypeptides which retain substantially the same or even exhibit a
reduction in the biological function or activity as the mature polypeptide encoded
by the polynucleotides described above.
The present invention further relates to polynucleotides that hybridize to the
herein above-described sequences. In this regard, the present invention especially
relates to polynucleotides which hybridize under stringent conditions to the herein
above-described polynucleotides. A preferred level of stringency is such that hybridization
will only occur if there is at least 85%, and preferably still 90%, and more preferably
95%, and even more preferably 97% identity between the sequences. The terms "identity"
and "similarity", as used herein, and as known in the art, are relationships between
two polypeptide sequences or two polynucleotide sequences, as determined by comparing
the sequences. In the art, identity also means the degree of sequence relatedness
between two polypeptide or two polynucleotide sequences as determined by the match
between two strings of such sequences. Both identity and similarity can be readily
calculate (Computational Molecular Biology, Lesk, A. M., ed., Oxford University
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part
I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton is Press, New
York, 1991). Methods commonly employed to determine identity or similarity between
two sequences include, but are not limited to those disclosed in Carillo, H., and
Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determine
identity are designed to give the largest match between the two sequences tested.
Methods to determine identity and similarity are codified in computer programs.
Typical computer program methods to determine identity and similarity between two
sequences include, GCG program package (Devereux, J., et al., Nucleic Acids Research
12(1):387 (1984)), BLASTP, BLASTN, FASTA and TFASTA (Atschul, S. F. et al., J.
Mol. Biol. 215:403 (1990)).
The terms "stringent conditions" or "stringent hybridization conditions" includes
reference to conditions under which a probe will hybridize to its target sequence,
to a detectably greater degree than other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will be different
in different circumstances. By controlling the stringency of the hybridization
and/or washing conditions, target sequences can be identified which are 100% complementary
to the probe (homologous probing). Alternatively, stringency conditions can be
adjusted to allow some mismatching in sequences so that lower degrees of similarity
are detected (heterologous probing). Generally, a probe is less than about 1000
nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration
is less than about 1.5M Na ion, typically about 0.01 to 1.0M Na ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°
C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C.
for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also
be achieved with the addition of destabilizing agents such as formamide. Exemplary
low stringency conditions include hybridization with a buffer solution of 30 to
35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash
in 1× to 2×SSC (20×SSC=3. 0M NaCl/0.3M trisodium citrate) at 50
to 55° C. Exemplary moderate stringency conditions include hybridization in
40 to 45% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.5× to
1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization
in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.1× SSC at
60 to 65° C.
Specificity is typically the function of post-hybridization washes, the
critical factors being the ionic strength and temperature of the final wash solution.
For DNA—DNA hybrids, the T
m can be approximated from the equation
of Meinkoth and Wahl,
Anal. Biochem., 138:267-284 (1984): T
.m=81.5°
C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent
cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA,
% form is the percentage of formamide in the hybridization solution, and L is the
length of the hybrid in base pairs. The T
m is the temperature (under
defined ionic strength and pH) at which 50% of a complementary target sequence
hybridizes to a perfectly matched probe. T
m is reduced by about 1°
C. for each 1% of mismatching; thus, T
m, hybridization and/or wash conditions
can be adjusted to hybridize to sequences of the desired identity. For example,
if sequences with 90% identity are sought, the T
m can be decreased 100°
C. Generally, stringent conditions are selected to be about 5° C. lower than
the thermal melting point (T
m) for the specific sequence and its complement
at a defined ionic strength and pH.
However, severely stringent conditions can utilize a hybridization and/or
wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T
m);
moderately stringent conditions can utilize a hybridization and/or wash at 6, 7,
8, 9, or 10° C. lower than the thermal melting point (T
m); low
stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14,
15, or 20° C. lower than the thermal melting point (T
m) Using the
equation, hybridization and wash compositions, and desired T
m, those
of ordinary skill will understand that variations in the stringency of hybridization
and/or wash solutions are inherently described. If the desired degree of mismatching
results in a T
m of less than 45° C. (aqueous solution) or 32°
C. (formamide solution) it is preferred to increase the SSC concentration so that
a higher temperature can be used. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview
of principles of hybridization and the strategy of nucleic acid probe assays",
Elsevier, N.Y. (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel,
et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
The teachings of all of the references cited throughout this specification are
incorporated herein by this reference to the extent that they are not inconsistent
with the teachings herein. It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in the art and
are to be included within the spirit and purview of this application and the scope
of the appended claims.
EXAMPLE 1
Transformation of
E.Coli with rtl Operon and Growth on Ribitol Medium
Materials and Methods
Initial work was done with clones of the operon for ribitol metabolism (Rbt)
from
Klebsiella pneumoniae, supplied by S. Turgot, Universität Osanbrtück.
From these, a ˜7.2 kb BamHI fragment was obtained from pFCK1, which contains
the entire Rbt operon, plus approximately 2.3 kb of sequences 3′ from the
operon. A ˜6.51 kb HindIII-BamHI fragment was obtained from pLTH9, which
lacks the first 720 bp from the repressor. Finally, a ˜3.98 kb ClaI fragment
was obtained from pLTH1, which lacks the repressor altogether, as well as any sequences
3′ to the operon. All bacteria were grown at 37° C. and shaken at 275-300 rpm.
The
K. pneumoniae fragments were released via enzymatic digestion as recommended
by the manufacturer (NEB, Beverly, Mass.), and blunted into the StuI site of pMECA
(Thomson and Parrott 1998). Following T4 DNA ligation with Fastlink ligase (Epicentre,
Madison, Wis.), pMECA was transformed via electroporation into
E. coli strain
DH10B (Life Technologies, Gaithersburg, Md.) and placed in 2B minimal broth as
recommended by BRL (Bethesda, Md.) supplemented with 2 g 1
-1 of ribitol
(=adonitol, Sigma, St. Louis), 50 mg 1
-1 each of L-leucine and L-isoleucine,
and 1 mg 1
-1 thiamine. The inorganic components of 2B medium are in
Table 1. All organic components were filter-sterilized. Only successful cloning
events of rbt were expected to result in bacterial growth, and successful growth
demonstrated that ribitol could be used to maintain a high-copy plasmid in an
E.
coli K-12 strain.
Next, the corresponding genes of the rtl operon were isolated from
E. Coli
strain C, which was obtained as stock number 3121 from the
E. coli Genetic
Stock Center at Yale University, and grown in 2B minimal broth supplemented with
2 g 1
-1 of ribitol. Total genomic DNA was isolated according to Syn
and Swarup (2000). The genomic DNA was subject to digestion by ClaI. Following
T4 DNA ligation into the corresponding site of pHEX3 (Heuel et al. 1997) and transformation
into DH10B, incubation took place in 5 ml of 2B minimal broth supplemented as described
previously. After 24 hours, 1 ml was placed in 25 ml of the same medium. As before,
only successful cloning events
