AU2001285459B2 - Methods for imparting desirable phenotypic traits, including drought, freeze, and high salt tolerance and methods for increasing seed production - Google Patents

Description

WO 02/16658 PCT/U S01/26039 METHODS AND COMPOSITIONS FOR MODULATING TELOMERASE REVERSE TRANSCRIPTASE (TERT) EXPRESSION CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. 119 this application claims priority to the filing dates of the United States Provisional Patent Application Serial Nos.

60/227,682 and 60/227,681, both filed August 24, 2000; the disclosures of which are herein incorporated by reference.

INTRODUCTION

Field of the Invention The field of this invention is the telomerase reverse transcriptase gene, specifically the regulation of the expression thereof.

Background of the Invention Telomeres, which define the ends of chromosomes, consist of short, tandemly repeated DNA sequences loosely conserved in eukaryotes. Human telomeres consist of many kilobases of (TTAGGG)n together with various associated proteins. Small amounts of these terminal sequences or telomeric DNA are lost from the tips of the chromosomes during S phase because of incomplete DNA replication. Many human cells progressively lose terminal sequence with cell division, a loss that correlates with the apparent absence of telomerase in these cells. The resulting telomeric shortening has been demonstrated to limit cellular lifespan.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA.

Human telomerase is made up of two components: an essential structural RNA (TER) (where the human component is referred to in the art as hTER); and a catalytic protein (telomerase reverse transcriptase or TERT) (where

I

depended on selective breeding of plants having desirable characteristics. Such selective breeding techniques, however, were often less than desirable as many plants had within them heterogeneous genetic complements that did not result in identical desirable traits to ¢C their parents.

[0004] Recently, advances in molecular biology have allowed mankind to Cmanipulate the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) 0 and the subsequent introduction of that genetic material into a plant or plant cells. Such technology has led to the development of plants with increased pest resistance, plants that n '10 are capable of expressing pharmaceuticals and other chemical, and plants that express beneficial traits. Advantageously such plants not only contain a gene of interest, but remain fertile and often desirably to pass the gene on to its progeny.

[0005] One particular area of interest of late has been the development of plants with improved production of seeds. Improving the yield in the production of seeds from cultivars, such as rice, canola, wheat, corn, and sunflower, can increase food production for animal and human consumption. Also, improved seed yield can have economic benefits by reducing the costs associated with producing seed for farming.

[0006] The yield of a plant crop may be improved by growing transgenic plants that are individually larger than the wild-type plant in vegetative and/or reproductive structure. It is known in the art that certain growth factors may be used to increase plant and/or plant flower size. Unfortunately, application of such growth factors is costly and time consuming, and do not necessarily substantially increase the yield of seeds from the plants. A need, therefore, exists for developing an improved method for increasing the yield of seeds from plants.

SUMMARY OF THE INVENTION [0006a] According to a first embodiment of the invention, there is provided a method of making a transgenic plant with increased resistance to the effects of an externally imposed stress selected from the group consisting of water deficit, drought, elevated salinity, and prolonged temperatures below 0°C, relative to non-transgenic wildtype plants, said method comprising: introducing into one or more cells of a plant an exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase in the cells to yield transformed cells; regenerating plants from the transformed cells to yield transgenic plants; and selecting a transgenic plant with increased resistance to the effects of an externally imposed stress selected from the group consisting of water deficit, drought, elevated salinity, and prolonged temperatures below O0C, relative to non-transgenic wildtype plants, thereby producing a transgenic plant with increased resistance to the effects of an externally imposed stress selected from the group consisting of water deficit, A627637spci drought, elevated salinity, and prolonged temperatures below 0°C, relative to non- Stransgenic wild-type plants.

[0006b] According to a second embodiment of the invention, there is provided a method of making a transgenic plant with enhanced ion accumulation in a plant vacuole of the transgenic plant, relative to non-transgenic wild-type plants, said method comprising: introducing into one or more cells of a plant an exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase in the cells to yield Stransformed cells; o0 regenerating plants from the transformed cells to yield transgenic plants; 00 and selecting a transgenic plant with enhanced ion accumulation in a plant O vacuole of the transgenic plant, relative to non-transgenic wild-type plants, thereby N producing a transgenic plant with enhanced ion accumulation in a plant vacuole of the is transgenic plant, relative to non-transgenic wild-type plants.

[0006c] Transgenic plants obtained by these methods, and transgenic seed and progeny thereof comprising the exogenous nucleic acid, are also provided.

[0006d] According to a third embodiment of the invention, there is provided a method for increasing production of seeds in plants, said method comprising: providing pollen from a transgenic plant, wherein the transgenic plant has been transformed with exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase to create a transgenic plant; fertilizing a plant with the pollen from the transgenic plant; and culturing the fertilized plant until the plant produces mature seeds.

[0006e] Transgenic plant seed comprising the exogenous gene, produced by such a method is also provided.

[0006f] According to a fourth embodiment of the invention, there is provided a transgenic plant with increased resistance to the effects of an externally imposed stress selected from the group consisting of water deficit, drought, elevated salinity and prolonged exposure to temperatures below 0°C, relative to non-transgenic wild-type plants, said transgenic plant comprising one or more transgenic plant cells, wherein the transgenic plant cells include an exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase in the transgenic plant cells.

[0006g] According to a fifth embodiment of the invention, there is provided a transgenic plant with enhanced capacity to accumulate ion species in a vacuole of the transgenic plant, relative to non-transgenic wild-type plants, said transgenic plant comprising one or more transgenic plant cells, wherein the transgenic plant cells include an exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase in the transgenic plant cells.

A627637spcci [0007] The transgenic plant of the invention is resistant to externally imposed 0stresses such as drought, prolonged exposure to temperatures below 0° C, and growth media high in salt content, where the growth media may be soil or water. Preferably, the cexogenous tonoplast driven H+ pump gene is operably linked to a double tandem enhancer of the 35S CaMV promoter. In addition, the present invention contemplates a seed produced by the transgenic plant of the invention, as well as a progeny plant from the seed of the plant of the invention.

O\ [0008] There is disclosed herein a transgenic plant containing a polynucleotide sequence comprising a multiplicity of exogenous tonoplast pyrophosphate driven H+ S 10 pump genes operably linked to a double tandem enhancer of the 35S CaMV promoter, C1 wherein the number of pyrophosphate driven H+ pump genes is sufficient to express a 0sufficient number of pyrophosphate driven H+ pumps on the vacuolar membranes to impart desirable phenotypic traits to the transgenic plant. Among these phenotypic traits are an ability to resist the effects of externally imposed stresses, wherein the externally imposed stresses to which the plant is resistant are exemplified by drought, prolonged exposure to temperatures below 00 C, and a growth medium high in salt content. The plant of this embodiment of the invention comprises exogenous nucleic acid that encodes AVPI or, alternatively, an homolog of AVP1. This homolog may be obtained from tobacco, bacteria, tomato or corn. The transgenic plants of this embodiment comprise an A VP1 gene in a construct designed to overexpress AVP1 or designed to down-regulate endogenous pyrophosphatase. In this construct, preferably the A VP1 is operably linked to a double tandem enhancer of a 35S CaMV promoter. Preferably, the A VP1 gene is derived from a wild type plant of the same species from which the transgenic plant is derived. Alternatively, the A VP1 gene is derived from a wild type plant of a different species from which the transgenic plant is derived.

[0009] As disclosed herein the transgenic plant is larger than a wild-type plant of the same species. Also, the invention contemplates a progeny plant of the transgenic plant, seeds produced by the transgenic plant, and a progeny plant grown from the seed.

[0010] There is disclosed herein a transgenic plant obtained by introducing into the genome of the plant exogenous nucleic acid that alters expression of vacuolar pyrophosphatase in the transgenic plant, as well as plant cells comprising exogenous nucleic acid that alters expression of vacuolar pyrophosphatase in the plant cell.

Preferably, the plant cells are selected from the group consisting of root cells and stem cells. Furthermore, the plant cells comprise exogenous nucleic acid that encodes the AVPI protein. Preferably, in the plant cells of the present invention, the exogenous nucleic acid that encodes AVP1 is present in a construct designed to overexpress AVP1 or designed to down-regulate endogenous pyrophosphatase. More preferably, the construct comprises the A VP1 gene operably linked to a chimeric promoter designed to overexpress AVPI. More preferably still, the A VP1 gene is operably linked to a double tandem enhancer of a 35S CaMV promoter. In this embodiment, the nucleotide encoding A627637speci AVPI is derived from a wild type plant of the same species from which the transgenic 0plant is derived, although the present invention also contemplates that the nucleotide N encoding AVP1 can be derived from a wild type plant of a different species from which c the transgenic plant is derived.

[0011] There is disclosed herein a method of making a transgenic plant that is Cc larger in size than its corresponding wild type plant comprising introducing into one or more cells of a plant a nucleotide sequence that alters expression of vacuolar pyrophosphatase in the plant to yield transformed cells, thereby producing a transgenic plant that is larger than its corresponding wild type plant. In this embodiment, the method tC 10 further comprises regenerating plants from the transformed cells to yield transgenic plants 00 C and selecting a transgenic plant that is larger than its corresponding wild type plant, 0thereby producing a transgenic plant which is larger than its corresponding wild type plant. Also encompassed by this embodiment of the present invention is a transgenic plant produced by this method.

[0012] There is disclosed herein a method of increasing the yield of a plant comprising introducing into one or more cells of a plant nucleic acid that alters expression of vacuolar pyrophosphatase in the plant to yield transformed cells, thereby increasing the yield of the plant. This method further comprises regenerating plants from the transformed cells to yield transgenic plants and selecting a transgenic plant that is larger than its corresponding wild type plant, thereby increasing the yield of the plant.

[0013] There is also disclosed herein a method of making a transgenic plant having increased flower size compared to its corresponding wild type plant comprising introducing into one or more cells of a plant nucleic acid that alters expression of vacuolar pyrophosphatase in the plant to yield transformed cells, thereby producing a transgenic plant having increased flower size compared to its corresponding wild type plant.

Preferably, the exogenous nucleic acid encodes AVP1. This embodiment of the invention also encompasses a transgenic plant produced by the method.

[0014] There is further disclosed herein a method of making a transgenic plant with increased biomass comprising introducing into one or more cells of a plant a nucleic acid construct that alters expression of vacuolar pyrophosphatase so as to increase vacuolar pyrophosphatase activity in the cell to yield transformed cells thereby producing a transgenic plant with increased biomass. This method further comprises regenerating plants from the transformed cells to yield transgenic plants and selecting a transgenic plant with increased biomass. Also encompassed by this embodiment of the present invention is a transgenic plant produced by the method.

[0015] There is still further disclosed herein a method of making a transgenic plant with an increased biomass over its corresponding wild type plant, wherein the increased biomass relates to an increase in the biomass of a plant part selected from the group consisting of leaves, stems, roots, seeds, flowers, and fruits; said method A627637spcci Scomprising introducing into one or more cells of a plant an exogenous nucleic acid that O alters expression of vacuolar pyrophosphatase so as to enhance the activity of the vacuolar pyrophosphatase in the plant to yield transformed cells, thereby producing a t transgenic plant with an increased biomass. This method, as provided by the present invention, further comprises regenerating plants from the transformed cells to yield transgenic plants and selecting a transgenic plant that is larger than its corresponding wild type plant, thereby producing a transgenic plant with an increased biomass. Preferably, according to this method, the exogenous nucleic acid encodes AVP1, or a homolog tI thereof. More preferably, the AVPI, or homolog thereof, is expressed from a construct designed to overexpress AVPI, or the homolog thereof. Preferably, this construct 00 comprises the A VP1 gene, or gene encoding a homolog of AVP1, wherein the gene is operably linked to a chimeric promoter designed to overexpress AVP1. More preferably, the AVP1 gene is operably linked to a chimeric promoter selected from the group Sconsisting of tissue specific promoters, constitutive promoters, inducible promoters and combinations thereof. In a most preferred embodiment, the A VP1 gene is operably linked to a tissue-specific promoter that promotes expression of AVP1 in pollen.

[0016] In one aspect of this disclosure, the AVP1 gene is operably linked to a double tandem enhancer of a 35S CaMV promoter. Preferably, the AVP1 gene, or homolog thereof, is derived from a wild type plant. Alternatively, the AVP1 gene, or homolog thereof, is derived from a transgenic plant. In yet another aspect of this embodiment, the A VP1 gene, or homolog thereof, is derived from a mutant plant. In one aspect, the transgenic plant is grown in soil, or is grown hydroponically. Also possible, is that a cell from transgenic plant is grown in culture. Also contemplated by this embodiment of the present invention is a transgenic plant produced by this method.

[0017] There is also disclosed herein a method of making a transgenic plant having increased root structure compared to its corresponding wild type plant comprising introducing into one or more cells of the plant an exogenous nucleic acid that alters expression of vacuolar pyrophosphatase so as to increase vacuolar pyrophosphatase activity in the plant to yield transformed cells, thereby producing a transgenic plant having increased root structure. According to this embodiment of the present invention, the exogenous nucleic acid encodes AVP1, or a homolog thereof. Also contemplated here is a transgenic plant produced by the method.

[0018] There is further disclosed herein a method for increasing production of seeds in plants comprising the steps of providing pollen from a first plant, wherein said first plant has been transformed with a tonoplast pyrophosphate driven H+ pump gene operably linked to a promoter to create a transgenic plant; fertilizing a second plant of the same species from which the first plant is derived with the pollen from the transgenic plant; and culturing the fertilized plant until the plant produces mature seeds. According to this method, the tonoplast pyrophosphatase driven H+ pump gene A627637speci 7 transformed into the first plant is exogenous. Also contemplated is that the second plant O is a transgenic plant or a wild type plant.

[0019] Preferably, the exogenous tonoplast pyrophosphate driven H+ pump gene is operably linked to a chimeric promoter. More preferably, the exogenous tonoplast pyrophosphate driven H+ pump gene is operably linked to a double tandem enhancer of c the 35S CaMV promoter. More preferably still, the exogenous tonoplast pyrophosphate driven H+ pump gene is operably linked to a double tandem enhancer of the 35S CaMV 0 promoter and is further operably linked to a multiple cloning site. In a most preferred embodiment, the exogenous tonoplast pyrophosphate driven H+ pump gene encodes AVP1. Also coming within this embodiment of the present invention is a plant seed Ci produced by the method; a progeny plant grown from the plant seed, wherein the first and 0 second plants used in the method are from the species A. thaliana. Alternatively, the first and second plants are from the species Nicotinia tabacum.

[0020] As disclosed herein the second plant has been transformed with a polynucleotide sequence comprising an exogenous tonoplast pyrophosphatase driven H+ pump gene operably linked to a promoter. Preferably, the polynucleotide sequence comprises an exogenous tonoplast pyrophosphatase driven H+ pump gene operably linked to a double tandem enhancer of the 35S CaMV promoter. Further, the polynucleotide sequence comprises an exogenous tonoplast pyrophosphatase driven H+ pump gene operably linked to a double tandem enhancer of the 35S CaMV promoter and further operably linked to a multiple cloning site. More preferably, the polynucleotide sequence comprises an exogenous tonoplast pyrophosphatase driven H+ pump gene operably linked to a double tandem enhancer of the 35S CaMV promoter and further operably linked to a heterologous coding sequence. This aspect of the present embodiment also contemplates a plant seed produced by the method and a progeny plant grown from the plant seed.

[0021] There is also disclosed herein pollen produced by a transgenic plant that has been transformed with a tonoplast pyrophosphatase driven H+ pump gene operably linked to a promoter. The pollen from these transgenic plants is more competent in fertilization, resulting in increased yield of seeds from the plants. Because most crops of interest are hermaphroditic, self-pollination of the transgenic plants by the more competent pollen will occur and will result in improved seed yield. The improved seed yield is demonstrated both by increased numbers of seeds and increased seed pod mass.

Increased seed yield can increase the production of products from cultivars such as rice, canola, wheat, corn and sunflower. Also, increased seed yield can decrease the cost of producing seeds to be used in crop production.

[0022] Other advantages of the present invention will become more readily apparent in view of the accompanying detailed description of the invention.

A627637speci BRIEF DESCRIPTION OF THE DRAWINGS [0023] So that those having ordinary skill in the art to which the subject invention appertains will more readily understand the subject invention, reference may be had to the drawings, wherein: [0024] Fig. IA is an overhead view of wild type (WT) and two independent transgenic lines (AVP1-1 and AVP1-2) after 10 days of water deprivation.

[0025] Fig. lB is an overhead view of the plants shown in Fig. IA after rewatering.

[0026] Fig. 2 is an overhead view of a representative wild type plant (WT) versus S 10 representative transgenic plants overexpressing AVPI (AVPI-1 and AVPI-2) after C exposure to 7 days of water deficit stress.

[0027] Figs. 3A, 3B and 3C are photomicrographs (magnification: times 40; bar CI length on photograph 2mm) of the root and root hairs of representative five day old seedlings obtained from representative WT, AVPI-1 transgenic and AVP1-2 transgenic plants grown parallel to the surface on vertical plant nutrient agar plates.

[0028] Fig. 4 is an immunoblot of membrane fractions isolated from wild type (WT) and two independent transgenic lines (AVP1-1 and AVP1-2) overexpressing AVP-1.

[0029] Fig. 5 is a drawing providing a perspective view of wild-type plants (WT) versus representative transgenic plants overexpressing AVP-1 (AVPI-1 and AVP1-2) grown in salty soil.

[0030] Fig. 6A is a graph showing accumulation of sodium ion in leaf tissue for wild-type plants (WT) versus representative transgenic plants overexpressing AVP-1 (AVPl-1 and AVP1-2).

[0031 Fig. 6B is a graph showing accumulation of potassium ion in leaf tissue for wild-type plants (WT) versus representative transgenic plants overexpressing AVP-1 (AVPI-1 and AVP1-2).

[0032] Fig. 6C is a graph showing the results of measurements of transport using vacuolar membrane vesicles derived from the AVP1 transgenic plant demonstrating that vacuoles from these plants have enhanced cation uptake capability.

[0033] Fig. 7 is a graph of the uptake of calcium into the 35SAVP-1 transgenic vacuolar membrane vesicles (squares) of AVPI -2 Fig. 5 versus calcium uptake into vesicles obtained from wild type (WT) of Fig. [0034] Figs. 8A and 8B are illustrations demonstrating the theorized mechanism for a higher accumulation of solids into vacuoles via a proton driven function versus that of WT vacuoles.

[0035] Fig. 9 is a graph showing seed yield for wild-type, AVPI-1 and AVP1-2 plants grown in a 16 hour light 8 hour dark cycle for two months.

[0036] Fig. 10A is a graph showing average number of seeds.

A627637spcci WO 02/16658 PCT/US01/26039 9 certain embodiments are sequences of substantially the same length as the specific nucleic acid identified above, where by substantially the same length is meant that any difference in length does not exceed about 20 number usually does not exceed about 10 number and more usually does not exceed about 5 number and have sequence identity to this sequence of at least about 90%, usually at least about 95% and more usually at least about 99% over the entire length of the nucleic acid.

Modulating TERT Expression The subject invention provides methods of modulating, including both enhancing and repressing, TERT expression. As such, methods of both increasing and decreasing TERT expression are provided. In practicing the subject invention, the repressive activity of the Myc Repeat region, particularly the target system that includes the Myc repeat region, e.g. the Myc repeat/Myc-Mad TERT expression repressive system, is modulated. Included are methods of either enhancing or inhibiting the TERT expression repressive activity of this target system.

In modulating TERT expression, the interaction between the Myc Repeat region and the one or more transacting factors of the target system with which it is acting, the Myc and Mad protein components of the Myc Repeat/Myc-Mad target system, is modified in a manner that achieves the desired change in TERT expression, enhancement or reduction. This target interaction can be modified directly or indirectly. An example of direct modification of this interaction is where the binding of the transacting factor(s), the Myc and/or Mad proteins, to the target Myc Repeat region is modified by an agent that directly changes how the transacting factor(s) binds to the Myc Repeat sequence, by occupying the DNA binding sites of the transacting factor, such as the E-box binding site of the Myc and/or Mad proteins (when combined with Max), by binding to the Myc Repeat region Eboxes thereby preventing the binding of this region to the transacting factor(s), etc. An example of indirect modification is modification/modulation of the target system repressive activity via disruption of a binding interaction between the transacting factor(s) and one or more cofactors (or further upstream in the WO 02/16658 PCT/US01/26039 chain of interactions) such that the repressive activity is modulated, by modification of the Myc Repeat sequence such that the repressive activity upon interaction with the transacting factors is modulated insertion or deletion of E-boxes), etc.

Enhancing TERT Expression Methods are provided for enhancing TERT expression. By enhancing TERT expression is meant that the expression level of the TERT coding sequence is increased by at least about 2 fold, usually by at least about 5 fold and sometimes by at least 25, 50, 100 fold and in particular about 300 fold or higher, as compared to a control, expression from an expression system that is not subjected to the methods of the present invention. Alternatively, in cases where expression of the TERT gene is so low that it is undetectable, expression of the TERT gene is considered to be enhanced if expression is increased to a level that is easily detectable.

In these methods, repression of TERT expression by the target system is inhibited. By inhibited is meant that the repressive activity of the target system with respect to TERT expression is decreased by a factor sufficient to at least provide for the desired enhanced level of TERT expression, as described above. Inhibition of the target system repression may be accomplished in a number of ways, where representative protocols for inhibiting this TERT expression repression are now provided.

One representative method of inhibiting repression of transcription is to employ double-stranded, duplex, oligonucleotide decoys for the transacting factor(s) of the target system, which bind to these transacting components and thereby prevent them from binding to their targets sequences, E-boxes, in the Myc Repeat region. These duplex oligonucleotide decoys have at least that portion of the sequence of the Myc Repeat site required to bind to the transacting factor(s), the Myc and/or Mad proteins, and thereby prevent their binding to the Myc Repeat region. In many embodiments, the subject decoy nucleic acid molecules include a sequence of nucleotides that is the same as a sequence found in SEQ ID NOs: 01 to 03. In other embodiments, the subject decoy nucleic acid WO 02/16658 PCT/US01/26039 11 molecules include a sequence of nucleotides that is substantially the same as or identical to a sequence found in SEQ ID NOs: 01 to 03; where the terms substantially the same as and identical thereto in relation to nucleic acids are defined below. In many embodiments, the length of these duplex oligonucleotide decoys ranges from about 5 to about 5000, usually from about to about 500 and more usually from about 10 to about 50 bases. In using such oligonucleotide decoys, the decoys are placed into the environment of the target system, resulting in de-repression of the transcription and expression of the TERT coding sequence. Oligonucleotide decoys and methods for their use and administration are further described in general terms in Morishita et al., Circ Res (1998) 82 (10):1023-8.

Instead of the above described decoys, other agents that disrupt binding of the target transacting factor, at least Mad in the specific Myc repeat/Myc-Mad target system described above, to the Myc Repeat region and thereby inhibit its expression repression activity may be employed. Other agents of interest include, among other types of agents, small molecules that bind to the transacting factor and inhibit its binding to the Myc repeat region.

Alternatively, agents that bind to the Myc Repeat sequence and inhibit its binding to transacting factor are of interest. Alternatively, agents that disrupt protein-protein interactions of the transacting factor with cofactors, e.g., cofactor binding, and thereby inhibit the transacting factor's binding to the target Myc Repeat region and consequently inhibit expression repression are of interest.

Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.

The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, WO 02/16658 PCT/US01/26039 12 pyrimidines, derivatives, structural analogs or combinations thereof. Such molecules may be identified, among other ways, by employing the screening protocols described below. Small molecule agents of particular interest include pyrrole-imidazole polyamides, analogous to those described in Dickinson et al., Biochemistry 1999 Aug 17;38(33):10801-7.

In yet other embodiments, expression of the transacting factor is inhibited. For example, in the specifically embodied Myc Repeat/Myc-Mad target system, inhibition of Mad expression is employed to achieve the desired increase in TERT expression, where this inhibition of Mad expression may be accomplished using any convenient means, including administration of an agent that inhibits Mad expression antisense agents), inactivation of the Mad gene, through recombinant techniques, etc. For example, antisense molecules can be used to down-regulate expression of the target repressor protein in cells. The anti-sense reagent may be antisense oligodeoxyribonucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted repressor protein, and inhibits expression of the targeted repressor protein. Antisense molecules inhibit gene expression through various mechanisms, e.g. by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule. Alternatively, the antisense molecule is a synthetic oligonucleotide.

Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be WO 02/16658 PCT/US01/26039 13 strong and selective inhibitors of gene expression (see Wagner et al. (1996), Nature Biotechnol 14:840-844).

A specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence.

Selection of a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993), supra, and Milligan et al., supra.) Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include phosphorothioate, 3'-S-5'-O-phosphorothioate, 3'-CH 2 -5'-O-phosphonate and Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural p-anomer. The 2'- OH of the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity.

Modification of the heterocyclic bases must maintain proper base pairing.

Some useful substitutions include deoxyuridine for deoxythymidine; 2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl- 2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.

WO 02/16658 PCT/US01/26039 14 As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene expression. Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman et a. (1995), Nucl. Acids Res.

23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of anti-sense ODN with a metal complex, e.g.

terpyridylCu(ll), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995), AppL. Biochem. BiotechnoL. 54:43-56.

In another embodiment, the transacting factor gene, the Mad gene, is inactivated so that it no longer expresses a functional repressor protein. By inactivated is meant that the target gene, coding sequence and/or regulatory elements thereof, is genetically modified so that it no longer expresses functional repressor protein. The alteration or mutation may take a number of different forms, through deletion of one or more nucleotide residues in the repressor region, through exchange of one or more nucleotide residues in the repressor region, and the like. One means of making such alterations in the coding sequence is by homologous recombination. Methods for generating targeted gene modifications through homologous recombination are known in the art, including those described in: U.S. Patent Nos. 6,074,853; 5,998,209 5,998,144; 5,948,653; 5,925,544; 5,830,698; 5,780,296; 5,776,744; 5,721,367; 5,614,396; 5,612,205; the disclosures of which are herein incorporated by reference.

The above described methods of enhancing TERT expression find use in a number of different applications. In many applications, the subject methods and compositions are employed to enhance TERT expression in a cell that endogenously comprises a TERT gene, e.g. for enhancing expression of hTERT in a normal human cell in which TERT expression is repressed. The target cell of these applications is, in many instances, a normal cell, e.g. a somatic cell. Expression of the TERT gene is considered to be enhanced if, consistent with the above description, expression is increased by at least about 2 fold, usually at least about 5 fold and often 25, 50, 100 fold, 300 fold or higher, as compared to a control, an otherwise identical cell not subjected WO 02/16658 PCT/US01/26039 to the subject methods, or becomes detectable from an initially undetectable state, as described above.

A more specific application in which the subject methods find use is to increase the proliferative capacity of a cell. The term "proliferative capacity" as used herein refers to the number of divisions that a cell can undergo, and preferably to the ability of the target cell to continue to divide where the daughter cells of such divisions are not transformed, they maintain normal response to growth and cell cycle regulation. The subject methods typically result in an increase in proliferative capacity of at least about 1.2 2 fold, usually at least about 5 fold and often at least about 10, 20, 50 fold or even higher, compared to a control. As such, yet another more specific application in which the subject methods find use is in the delay of the occurrence of cellular senescence. By practicing the subject methods, the onset of cellular senescence may be delayed by a factor of at least about 1.2 2 fold, usually at least about 5 fold and often at least about 10, 20, 50 fold or even higher, compared to a control.

Methods of Inhibiting TERT Expression As mentioned above, also provided are methods for inhibiting TERT expression, by enhancing repression of TERT expression by the target system and thereby inhibiting TERT expression. In such methods, the amount and/or activity of transacting factor, Mad, is increased so as to enhance repression of TERT expression by the target system. A variety of different protocols may be employed to achieve this result, including administration of an effective amount of the transacting factor or analog/mimetic thereof, an agent that enhances expression of the transacting factor or an agent that enhances the activity of the transacting factor.

As such, nucleic acid compositions that encode the transacting factor find use in situations where one wishes to enhance the activity of transacting factor in a host. The repressor protein genes, gene fragments, or the encoded proteins or protein fragments are useful in gene therapy to treat disorders in which inhibition of TERT expression is desired, including those applications described in greater detail below. Expression vectors may be used to WO 02/16658 PCT/US01/26039 16 introduce the gene into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.

The gene or protein may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles.

Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992), Anal Biochem 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992), Nature 356:152-154), where gold microprojectiles are coated with the DNA, then bombarded into skin cells.

Therapeutic Applications of TERT Expression Modulation The methods find use in a variety of therapeutic applications in which it is desired to modulate, increase or decrease, TERT expression in a target cell or collection of cells, where the collection of cells may be a whole animal or portion thereof, tissue, organ, etc. As such, the target cell(s) may be a host animal or portion thereof, or may be a therapeutic cell (or cells) which is to be introduced into a multicellular organism, a cell employed in gene therapy. In such methods, an effective amount of an active agent that modulates TERT expression, enhances or decreases TERT expression as desired, is administered to the target cell or cells, by contacting the cells with the agent, by administering the agent to the animal, etc. By effective amount is meant a dosage sufficient to modulate TERT expression in the target cell(s), as desired.

In the subject methods, the active agent(s) may be administered to the targeted cells using any convenient means capable of resulting in the desired 17 O Berkowitz, P. Pier, Plant Physiol. 89, 1358-1365 (1989)), it is hypothesized (but the N1 invention is not hereby limited by such theory) that the increased drought resistance of the AVP-I transgenic plants may be a consequence of their higher vacuolar concentration of potassium that results in a increased water retention capability. Laboratory tests appear to c 5 confirm this.

[0068] Five wild-type plants (WT) and two AVP-1 overexpressing transgenic t lines (AVPI-1 and AVPl-2) were grown on soil in a 10 hour light/dark cycle. Plants tt were watered with a diluted nutrient solution (1/8 MS salts) for six weeks and subsequently watered with a diluted nutrient solution supplemented with NaCl. The 0o concentration of NaCl began with 100 mM and was increased every four days by 100 mM. The photograph in Fig. IA corresponds to plants at the tenth day in the presence of 300 mM NaCl. Fig. 3 illustrates that the two AVP-1 plant types (AVPI-1 and AVP1-2) were significantly hardier in salty soil as compared to wild-type plants. The fact that genetically engineered Arabidopsis thaliana plants that overexpress either AVP1 (the pyrophosphate-energized vacuolar membrane proton pump, this work) or AtNHX1 (the Na+/H 4 antiporter, (Apse, at al., Science, 285:1256-1258 (1999)) and this work) are capable of growing in the presence of high NaCI concentrations strongly supports the strategy described herein. A double transgenic plant would be expected to demonstrate a further enhanced salt-tolerant phenotype. These Arabidopsis thaliana transporters or their counterparts may perform similar function in important agricultural crops. Fig. 5 is a drawing of wild type plants (WT) versus representative transgenic plants overexpressing AVPI (AVPI-1 and AVP1-2) grown in salty soil.

[0069] A Working Model of Cation Homeostasis in Plant Organelles While the present invention is not limited to any particular hypothesis, the present inventors have developed a working model for cation homeostasis in plant cells which can explain the observed results with respect to the transgenic plants disclosed herein.

[0070] In plants, most of the transport processes are energized by the primary translocation of protons. H -translocating pumps located at the plasma membrane and tonoplast translocated H from the cytosol to extracellular and vacuolar compartments, respectively (Rea, et al., Tonoplast Adenosine Triphosphate and inorganic Pyrophosphatase. In: Methods Plant Biochem., pp. 385-405, Academic Press Limited, London (1990)). The plant tonoplast contains two H 4 -translocating pumps; the V- ATPase and the inorganic pyrophosphatase or V-PPase. Their action results in luminal acidification and the establishment of a H electrochemical potential gradient across the A627637speci tonoplast (Davies, et al., The Bioenergetics of Vacuolar H' Pumps. In: Plant SVacuole, pp. 340-363, Leigh, Sanders, D. Academic Press, San Diego (1997)). The vacuolar membrane is implicated in a broad spectrum of physiological processes that include cytosolic pH stasis, compartmentation of regulatory Ca 2 sequestration of toxic ions such as Na turgor regulation, and nutrient storage and retrieval. The vacuole constitutes 40 to 99% of the total intracellular volume of a mature plant cell. The vacuolar proton pumping pyrophosphatase is a universal and abundant t component of plant tonoplast capable of generating a steady-state trans-tonoplast H electrochemical potential similar or greater than the one generated by the V-ATPase (Rea, o0 et al., Tonoplast Adenosine Triphosphate and Inorganic Pyrophosphatase. In: SMethods Plant Biochem., pp. 385-405, Academic Press Limited, London (1990)).

Pyrophosphate (PPi) is a by-product in the activation or polymerization steps of a wide range of biosynthetic pathways and in plants serves as an alternative energy donor to ATP for sucrose mobilization via sucrose synthetase, for glycolysis via PPi: fructose-6phosphate phosphotransferase and for tonoplast energization via the vacuolar proton pumping pyrophosphatase (Stitt, Bot. Acta 111:167-175 (1998)).

[0071] Most of intracellular organelles, including clathrin-coated vesicles, endosomes, Golgi membranes and vacuoles have acidic interiors (Xie, X. et al., J.

Biol.. Chem., 264:18870-18873 (1989)). This acidification is mediated by a protontranslocating electrogenic ATPase and in plant vacuoles also via a pyrophosphate-driven proton pump V-PPase (Davies, et al., The Bioenergetics of Vacuolar H Pumps. In: Leigh Sanders, (eds) The Plant Vacuole, pp. 340-363, Academic Press, San Diego (1997); Zhen, et al., "The Molecular and Biochemical Basis of Pyrophosphate-Energized Proton Translocation at the Vacuolar Membrane Academic Press Limited (1997)). There exists a requirement of anion transport to maintain net electroneutrality (al-Awqati, Curr. Opin. Cell. Biol., 7:504-508 (1995)).

[0072] Two transgenic lines of Arabidopsis thaliana were analyzed, A VPl-1 and A VPI-2. Each line contains extra copies of the 35S::A VPI gene inserted at a single chromosomal location. Analysis of AVPI protein levels in membrane fractions isolated from shoots shows that these transgenic plants express more AVPI protein than does the wild type (AVPI-I, 1.6 fold and AVPJ-2, 2.4 fold increase over wild type, P-value 0.0005) (Fig. 4) as determined from four independent immunoblots. The differences between these transgenic plants could be due to the number of copies of A VPI inserted into the genome or the sites of insertion. The transgenic plants overexpressing AVPI are more salt tolerant than wild type plants (Figs, 2 and Plants from both A VPl-1 and A627637spcci 19 A VPI-2 transgenic lines grow well in the presence of up to 250 mM NaCl whereas wild i type plants grow poorly end exhibit chlorosis. After 10 days in these conditions wild type plants die, whereas the transgenic plants continue to grow well.

[0073] The enhanced tolerance to salinity and drought in transgenic plants with s increased levels of AVPI is most easily explained by an enhanced uptake of toxic cations such as sodium into the vacuole. Presumably, the greater AVP1 activity provides 0increased H+ to drive the secondary active uptake of cations into the lumen of the vacuole S(Fig. 6C). If so, there must be a compensatory transport of anions to maintain 00 electroneutrality. The resulting elevated vacuolar solute content would confer greater S 10 water retention, permitting plants to survive under conditions of low soil water potentials.

0Furthermore, at high Na concentrations, the increased H gradient could also enhance the driving force for AtNHX-1-mediated Na+/H exchange, thereby contributing to the Na sequestration into the vacuole of A VP1 transgenic plants. Presumably, any toxic effects intrinsic to Na are mitigated by this sequestration in the vacuole. This scenario predicts that a transgenic plant engineered to overexpress both, the AVPI H+-pump and the AtNHX1 Na+/H antiporter would tolerate even higher NaCI stresses than AVP1 and AtNHXI single transgenic plants.

[0074] Figs. 6A and 6B show a graph of Na and K content, respectively of wildtype plants (WT) versus representative transgenic plants overexpressing AVP-1 and grown in salty soil. Five wild-type plants (WT) and two AVP-1 overexpressing transgenic lines and were grown on soil in a 10 hour light/dark cycle. Plants were watered with a diluted nutrient solution (1/8 MS salts) for six weeks and subsequently watered with a diluted nutrient solution supplemented with NaCI. The concentration of NaCI began with 100 mM and was increased every four days by 100 mM. The data correspond to plants at the tenth day in the presence of 300 mM NaCI. Parts of the plant above ground were harvested after 24 hours in the presence of 200 mM NaCl and their fresh weigh measured. After 48 hours at 75 0 C, the dry weight was measured. Na and K+ content was determined by atomic absorption. Values in the graphs of Figs. 6A and 6B are the mean SE (n As can be seen from the graphs Na' and K+ content in the transgenic lines and was significantly higher than that of wild-type counterparts.

[0075] Figs. 6C and 7 are graphs of the uptake of calcium into the 35SAVP-1 transgenic vacuolar membrane vesicles (closed squares) of AVPl-2 versus calcium uptake into vesicles obtained from wild type Wild-type plants (closed squares in Fig. 6C; open squares in Fig. 7) and transgenic plants from line AVPI-2 were grown A627637spcci r hydroponically for nine weeks on a 10 hour light cycle. Vacuolar membrane vesicles were added to buffer containing 250 mM sorbitol, 25 mM BTP-Hepes pH 8.0, 50 mM KCI, 1.5 nM MgSO 4 and 10 AM Ca This mix was incubated at 20 0 C for 10 minutes before adding 200 /M PPi to trigger the reaction. Ca ionophore A23187 was added to a final concentration of 5 Ag/ml to dissipate the Ca" gradient. Aliquot (200 pl) were filtered at the indicated times and washed with cold buffer as described As is evidenced by the graph, the transgenic plants from line 2' have greater calcium uptake t than wild-type plants.

00 [0076] The above data are consistent with the hypothesis that transgenic plants to overexpressing A VP-1 have an enhanced H pumping capability at their tonoplast and that San enhanced H supply results in greater ion accumulation in the vacuole through the action of H+-driven ion transporters. To further support this theory, Ca" uptake capability of wild type and transgenic vacuolar membrane vesicles was determined.

[0077] It is well documented that Ca enters the plant vacuole via a Ca+/H antiporter S. Schumaker, H. Sze, Plant Physiol. 79, 1111-1117 (1985)). Furthermore, the genes encoding the Arabidopsis thaliana Ca+/H' antiporters CAX1 and CAX2 have been isolated and characterized D. Hirschi, Zhen, K. W. Cunningham, P. A.

Rea, G. R. Fink, Proc. Natl. Acad. Sci. USA 93, 8782-8786 (1996)). Fig. 7 shows that Ca" uptake in the 35SA VP-1 transgenic vacuolar membrane vesicles is 36% higher than it is in vesicles obtained from wild type. Application of the Ca ionophore A23187 lowered the 45Ca counts to background levels demonstrating the tightness of the vesicles (see Fig. 6C) S. Schumaker, H. Sze, Plant Physiol. 79, 1111-1117 (1985)).

[0078] While not limited by such theory, a model consistent with the enhanced drought and freeze tolerance of the transgenic plants overexpressing the A VP-1 gene is depicted in Figs. 8A and 8B. The model depicts how an increase in the number of AVP-1 pumps in the vacuole of transgenic plants can provide more H+ that will permit the secondary transporters to import greater amounts of cations into the lumen of the vacuoles. Higher amounts of cations confer a greeter osmotic pressure that leads to a greater water retention capability endowing plants to withstand low soil water potentials.

[0079] The present invention relates, in one aspect, to pollen produced by a transgenic plant transformed with a tonoplast pyrophosphatase driven H+ pump gene operably linked to a promoter. In a second aspect, the invention relates to methods for increasing the production of seeds in plants using pollen from a transgenic plant A627037speci 21 transformed with a tonoplast pyrophosphatase driven H+ pump gene operably linked to a promoter.

S[0080] Transgenic plants that overexpress a vacuolar proton-pumping pyrophosphatase, such as for example AVP 1, also produce an increased yield of seeds.

Referring to Fig. 9, the seed yield, as expressed by the weight of seeds produced, is higher for AVPI-1 transgenic plants as compared to wild type plants. This increased seed yield 0 is a result of the pollen from the transgenic plant having an enhanced ability to fertilize, referred to herein as fertilization competence.

00 [0081] To demonstrate that the improved seed yield is a result of the improved to fertilization competence of the pollen from the transgenic plant, the pollen from wild type N, Arabidosis thaliana plants was used for pollination of two lines of transgenic Arabidosis thaliana plants transformed to overexpress AVP 1 (these two lines of transgenic plants are referred to herein as AVP 1-1 and AVP Referring to Figs. 10A and 10B, the transgenic plants pollinated with pollen from wild type plants produced an average of between about 15 and 20 seeds, with an average seed pod mass of between about 2.5 and 3 milligrams. These results were compared to the seed yield obtained when pollen from the two lines of Arabidosis thaliana transgenic plants was used to pollinate wild type Arabidosis thaliana plants. Referring again to Figs. 10A and 10B, the wild type plants fertilized with transgenic pollen produced an average of between about 30 and 35 seeds, with an average seed pod mass of between about 4 and 5 milligrams.

[0082] These results demonstrate that pollen from transgenic plants transformed with a tonoplast pyrophosphatase-driven H' pump gene is capable of causing improved seed yield in plants fertilized with the transgenic pollen. To further illustrate, wild type plants that are fertilized with transgenic pollen also produce an increased yield of seeds, while transgenic plants fertilized with wild type pollen do not. These results clearly indicate that it is the pollen from the transgenic plant, and not the female reproductive organs of the transformed plant, that causes improved seed yield.

[0083] Similar results have also been observed in other plant species transformed to overexpress a vacuolar proton-pumping pyrophosphatase. Referring to Fig. 11, the volume of seeds produced by wild type tobacco plants is compared to the seed pod volume produced in transgenic tobacco plants transformed to overexpress a vacuolar proton-pumping pyrophosphatase. The volume of five seed pods from each plant was weighed. For the wild type tobacco plants, the volume of seeds in five pods was between about 0.5 milliliters and 0.8 milliliters. For the three lines of transgenic tobacco plants tested, the volume of seeds in five pods was between about 1.2 milliliters and 1.4 A627637spcct 22 r milliliters. The transgenic tobacco lines were crossed and the volume of five seed pods was measured. The volume of five seed pods from the crossed lines of tobacco plants remained between about 1.2 milliliters and 1.4 milliliters.

[0084] These results further demonstrate that it is the pollen from the transgenic plants that improve seed yield. All three lines of transgenic tobacco plants had substantially greater seed pod volume than wild type plants. In addition, when the three lines of transgenic tobacco plants were crossed, the seed pod volume was about the same t as the seed pod volume of the uncrossed transgenic line, and much greater than the seed 0t pod volume from the wild type line.

00 [0085] Most food plants of interest are hermaphroditic and will self-pollinate.

Transgenic plants of this type that have been transformed to overexpress a vacuolar proton-pumping pyrophosphatase will themselves produce increased seed yields as a result of the improved fertilization competence of their pollen.

[0086] In another aspect of the present invention, which is especially useful for plant species which do not self-pollinate, pollen is provided from a transgenic plant transformed with a tonoplast pyrophosphatase driven H' pump gene operably linked to a promoter. Wild type female flowers are fertilized by pollen from wild type plants (Figs.

12A; 13A) or pollen from transgenic plants (Figs. 12B; 13B).

[0087] After the wild type plant has been fertilized, the plant is cultured until the wild type plant produces mature seeds. The mature seeds are harvested from the wild type plant after they reach maturity. This increase in seed yield is a result of the improved competence of the pollen from the transgenic plant in fertilization.

[0088] In another embodiment of the present invention, the pollen from a transgenic plant transformed with a tonoplast pyrophosphatase driven H+ pump gene operably linked to a promoter is used to fertilize a transgenic plant which has also been transformed with a tonoplast pyrophosphatase driven H+ pump gene operably linked to a promoter. After the transgenic plant has been fertilized, the plant is cultured until it produces mature seeds. The mature seeds are harvested from the transgenic plant after they reach maturity.

A62763 7 spcci WO 02/16658 PCT/US01/26039 23 meant to increase the time during which the animal is alive, where the increase is generally at least 1 usually at least 5% and more usually at least about 10 as compared to a control.

As indicated above, instead of a multicellular animal, the target may be a cell or population of cells which are treated according to the subject methods and then introduced into a multicellular organism for therapeutic effect. For example, the subject methods may be employed in bone marrow transplants for the treatment of cancer and skin grafts for burn victims. In these cases, cells are isolated from a human donor and then cultured for transplantation back into human recipients. During the cell culturing, the cells normally age and senesce, decreasing their useful lifespans. Bone marrow cells, for instance, lose approximately 40 of their replicative capacity during culturing.

This problem is aggravated when the cells are first genetically engineered (Decary, Mouly et al. Hum Gene Ther 7(11): 1347-50, 1996). In such cases, the therapeutic cells must be expanded from a single engineered cell. By the time there are sufficient cells for transplantation, the cells have undergone the equivalent of 50 years of aging (Decary, Mouly et al. Hum Gene Ther 8(12): 1429-38, 1997). Use of the subject methods spares the replicative capacity of bone marrow cells and skin cells during culturing and expansion and thus significantly improves the survival and effectiveness of bone marrow and skin cell transplants. Any transplantation technology requiring cell culturing can benefit from the subject methods, including ex vivo gene therapy applications in which cells are cultured outside of the animal and then administered to the animal, as described in U.S. Patent Nos. 6,068,837; 6,027,488; 5,824,655; 5,821,235; 5,770,580; 5,756,283; 5,665,350; the disclosures of which are herein incorporated by reference.

Treatment of Disease Conditions by Decreasing TERT Expression As summarized above, also provided are methods for enhancing repression of TERT expression, where by enhancement of TERT expression repression is meant a decrease in TERT expression by a factor of at least about 2 fold, usually at least about 5 fold and more usually at least about fold, as compared to a control. Methods for enhancing Myc Repeat region WO 02/16658 PCT/US01/26039 24 mediated repression of TERT expression find use in, among other applications, the treatment of cellular proliferative disease conditions, particularly abnormal cellular proliferative disease conditions, including, but not limited to, neoplastic disease conditions, cancer. In such applications, an effective amount of an active agent, a transacting factor, analog or mimetic thereof, (such as Mad) a vector encoding the same or active fragment thereof, an agent that enhances endogenous transacting factor activity, an agent that enhances expression of the transacting factor, etc., is administered to the subject in need thereof. Treatment is used broadly as defined above, to include at least an amelioration in one or more of the symptoms of the disease, as well as a complete cessation thereof, as well as a reversal and/or complete removal of the disease condition, cure. Methods of treating disease conditions resulting from unwanted TERT expression, such as cancer and other diseases characterized by the presence of unwanted cellular proliferation, are described in, for example, U.S. Patent Nos. 5,645,986; 5,656,638; 5,703,116; 5,760,062; 5,767,278; 5,770,613; and 5,863,936; the disclosures of which are herein incorporated by reference.

NUCLEIC ACID COMPOSITIONS Also provided by the subject invention are nucleic acid compositions, where the compositions are present in other than their natural environment, are isolated, recombinant, etc., that include a Myc Repeat domain/region, as described above. In other embodiments, the subject nucleic acids have a sequence that is substantially the same as, or identical to, the Myc Repeat sequences as described above, SEQ ID NOs: 01 to 03. A given sequence is considered to be substantially similar to this particular sequence if it shares high sequence similarity with the above described specific sequences, e.g. at least 75% sequence identity, usually at least 90%, more usually at least 95% sequence identity with the above specific sequences.

Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence WO 02/16658 PCT/US01/26039 analysis are known in the art, such as BLAST, described in Altschul et aL (1990), J. MoL Biol. 215:403-10 (using default settings, i.e. parameters w=4 and T=17). Of particular interest in certain embodiments are nucleic acids of substantially the same length as the specific nucleic acid identified above, where by substantially the same length is meant that any difference in length does not exceed about 20 number usually does not exceed about number and more usually does not exceed about 5 number and have sequence identity to this sequence of at least about 90%, usually at least about 95% and more usually at least about 99% over the entire length of the nucleic acid.

Also provided are nucleic acids that hybridize to the above described nucleic acid under stringent conditions. An example of stringent hybridization conditions is hybridization at 500c or higher and 0.1xSSC (15 mM sodium mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42 0 C in a solution: 50 formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 Vtg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least about 90% as stringent as the above specific stringent conditions. Other stringent hybridization conditions are known in the art and may also be employed to identify nucleic acids of this particular embodiment of the invention.

In many embodiments, the above described nucleic acid compositions include the Myc repeat domain region but do not include all of the components of the TERT genomic sequence, all of the other intron/exon regions of the TERT genomic sequence. In these embodiments, the subject nucleic acids include no more than about 90 number usually no more than about number and more usually no more than about 75 number where in many embodiments the subject nucleic acids include less than about 50 number sometimes less than about 40 number and sometimes less than about number of the total sequence of the TERT genomic sequence. In certain WO 02/16658 PCT/US01/26039 26 embodiments, the length of the subject nucleic acids ranges from about 5 to about 5000 bases, sometimes from about 10 to about 2500 bases and usually from about 10 to about 1000 bases.

The above described nucleic acid compositions find use in a variety of different applications, including the preparation of constructs, vectors, expression systems, etc., as described more fully below, the preparation of probes for the Myc Repeat sequence in non-human animals, non-human Myc Repeat homologs, and the like. Where the subject nucleic acids are employed as probes, a fragment of the provided nucleic acid may be used as a hybridization probe against a genomic library from the target organism of interest, where low stringency conditions are used. The probe may be a large or small fragment, generally ranging in length from about 10 to 100 nt, usually from about 15 to 50 nt. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50'C and 6xSSC (0.9 M sodium chloride/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in IxSSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequence identity may be determined by hybridization under stringent conditions, for example, at 50 0 C or higher and 0.1xSSC (15 mM sodium chloride/01.5 mM sodium citrate). Nucleic acids having a region of substantial identity to the provided nucleic acid sequences bind to the provided sequences under stringent hybridization conditions. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related sequences.

The subject nucleic acids are isolated and obtained in substantial purity, generally as other than an intact chromosome. As such, they are present in other than their naturally occurring environment. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a Myc repeat region or fragment thereof, generally being at least about usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

The subject nucleic acids may be produced using any convenient protocol, including synthetic protocols, those where the nucleic acid is synthesized by a sequential monomeric approach via phosphoramidite WO 02/16658 PCT/US01/26039 27 chemistry); where subparts of the nucleic acid are so synthesized and then assembled or concatamerized into the final nucleic acid, and the like. Where the nucleic acid of interest has a sequence that occurs in nature, the nucleic acid may be retrieved, isolated, amplified etc., from a natural source using conventional molecular biology protocols.

Also provided are nucleic acid compositions that include a modified or altered Myc Repeat region, where the site includes one or more deletions or substitutions as compared to the above specific Myc Repeat region. The subject nucleic acids of this embodiment that include a deletion (or substitution) in all or a portion of the Myc repeat may be present in the genome of a cell or animal of interest, as a "knockout" deletion in a transgenic cell or animal, where the cell or animal initially has this region, or may be present in an isolated form. A "knockout" animal could be produced from an animal that originally has the subject Myc Repeat using the sequences flanking specific Myc Repeat regions described here and the basic "knockout" technology known to those skilled in the art e.g. see U.S. Patent 5,464,764 to Capecchi.

Also provided are constructs comprising the subject nucleic acid compositions, those that include the Myc Repeat or those that include a deletion in the Myc Repeat region, inserted into a vector, where such constructs may be used for a number of different applications, including propagation, screening, genome alteration, and the like, as described in greater detail below. Constructs made up of viral and non-viral vector sequences may be prepared and used, including plasmids, as desired. The choice of vector will depend on the particular application in which the nucleic acid is to be employed. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture, for use in screening assays. Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art.

Many such vectors are available commercially. To prepare the constructs, the partial or full-length nucleic acid is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector.

Alternatively, the desired nucleotide sequence can be inserted by homologous WO 02/16658 PCT/US01/26039 28 recombination in vivo. Typically this is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence.

Regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example.

Additional examples of nucleic acid compositions that include the Myc Repeat are polymers, e.g. a double stranded DNA molecules, that mimic the Myc repeat site as described above.

Also provided are expression cassettes, vectors or systems that find use in, among other applications, screening for agents that modulate, e.g., inhibit or enhance the repressive activity of the region, as described in greater detail below; and/or to provide for expression of proteins under the control of the expression regulation mechanism of the TERT gene. By expression cassette or system is meant a nucleic acid that includes a sequence encoding a peptide or protein of interest, a coding sequence, operably linked to a promoter sequence, where by operably linked is meant that expression of the coding sequence is under the control of the promoter sequence. The expression systems and cassettes of the subject invention comprise a Myc Repeat region that, in the presence of the other target system components, the Mad/Myc components, of the target expression repression system, can modulate expression of a coding sequence to which it is operably linked.

As indicated above, expression systems comprising the subject regions find use in applications where it is desired to control expression of a particular coding sequence using the TERT transcriptional mechanism. In such applications, the expression system further includes the coding sequence of interest operably linked to the Myc Repeat element. The expression system is then employed in an appropriate environment to provide expression or nonexpression of the protein, as desired, in an environment in which telomerase is expressed, a Hela cell, or in an environment in which telomerase is not expressed, an MRC5 cell. Alternatively, the expression system may be used in an environment in which telomerase expression is inducible, by adding to the system an additional agent that turns on telomerase expression.

WO 02/16658 PCT/US01/26039 29 The above applications of the subject nucleic acid compositions are merely representative of the diverse applications in which the subject nucleic acid compositions find use.

GENERATION OF ANTIBODIES Also provided are methods of generating antibodies, monoclonal antibodies. In one embodiment, the blocking or inhibition, either directly or indirectly as described above, of the TERT expression repressive activity of the Myc Repeat region is used to immortalize cells in culture, by enhancing telomerase expression. Exemplary of cells that may be used for this purpose are non-transformed antibody producing cells, e.g. B cells and plasma cells which may be isolated and identified for their ability to produce a desired antibody using known technology as, for example, taught in U.S.

patent 5,627,052. These cells may either secrete antibodies (antibodysecreting cells) or maintain antibodies on the surface of the cell without secretion into the cellular environment. Such cells have a limited lifespan in culture, and are usefully immortalized by upregulating expression of telomerase using the methods of the present invention.

Because the above described methods are methods of increasing expression of TERT and therefore increasing the proliferative capacity and/or delaying the onset of senescence in a cell, they find applications in the production of a range of reagents, typically cellular or animal reagents. For example, the subject methods may be employed to increase proliferation capacity, delay senescence and/or extend the lifetimes of cultured cells.

Cultured cell populations having enhanced TERT expression are produced using any of the protocols as described above, including by contact with an agent that inhibits repressor region transcription repression and/or modification of the repressor region in a manner such that it no longer represses TERT coding sequence transcription, etc.

WO 02/16658 PCT/US01/26039 The subject methods-find use in the generation of monoclonal antibodies. An antibody-forming cell may be identified among antibodyforming cells obtained from an animal which has either been immunized with a selected substance, or which has developed an immune response to an antigen as a result of disease. Animals may be immunized with a selected antigen using any of the techniques well known in the art suitable for generating an immune response. Antigens may include any substance to which an antibody may be made, including, among others, proteins, carbohydrates, inorganic or organic molecules, and transition state analogs that resemble intermediates in an enzymatic process. Suitable antigens include, among others, biologically active proteins, hormones, cytokines, and their cell surface receptors, bacterial or parasitic cell membrane or purified components thereof, and viral antigens.

As will be appreciated by one of ordinary skill in the art, antigens which are of low immunogenicity may be accompanied with an adjuvant or hapten in order to increase the immune response (for example, complete or incomplete Freund's adjuvant) or with a carrier such as keyhole limpet hemocyanin (KLH).

Procedures for immunizing animals are well known in the art. Briefly, animals are injected with the selected antigen against which it is desired to raise antibodies. The selected antigen may be accompanied by an adjuvant or hapten, as discussed above, in order to further increase the immune response.

Usually the substance is injected into the peritoneal cavity, beneath the skin, or into the muscles or bloodstream. The injection is repeated at varying intervals and the immune response is usually monitored by detecting antibodies in the serum using an appropriate assay that detects the properties of the desired antibody. Large numbers of antibody-forming cells can be found in the spleen and lymph node of the immunized animal. Thus, once an immune response has been generated, the animal is sacrificed, the spleen and lymph nodes are removed, and a single cell suspension is prepared using techniques well known in the art.

Antibody-forming cells may also be obtained from a subject which has generated the cells during the course of a selected disease. For instance, antibody-forming cells from a human with a disease of unknown cause, such as rheumatoid arthritis, may be obtained and used in an effort to identify WO 02/16658 PCT/US01/26039 31 antibodies which have an effect on the disease process or which may lead to identification of an etiological agent or body component that is involved in the cause of the disease. Similarly, antibody-forming cells may be obtained from subjects with disease due to known etiological agents such as malaria or AIDS. These antibody forming cells may be derived from the blood or lymph nodes, as well as from other diseased or normal tissues. Antibody-forming cells may be prepared from blood collected with an anticoagulant such as heparin or EDTA. The antibody-forming cells may be further separated from erythrocytes and polymorphs using standard procedures such as centrifugation with Ficoll-Hypaque (Pharmacia, Uppsula, Sweden). Antibodyforming cells may also be prepared from solid tissues such as lymph nodes or tumors by dissociation with enzymes such as collagenase and trypsin in the presence of EDTA.

Antibody-forming cells may also be obtained by culture techniques such .as in vitro immunization. Briefly, a source of antibody-forming cells, such as a suspension of spleen or lymph node cells, or peripheral blood mononuclear cells are cultured in medium such as RPMI 1640 with 10% fetal bovine serum and a source of the substance against which it is desired to develop antibodies. This medium may be additionally supplemented with amounts of substances known to enhance antibody-forming cell activation and proliferation such as lipopolysaccharide or its derivatives or other bacterial adjuvants or cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6, GM-CSF, and IFN- .gamma.. To enhance immunogenicity, the selected antigen may be coupled to the surface of cells, for example, spleen cells, by conventional techniques such as the use of biotin/avidin as described below.

Antibody-forming cells may be enriched by methods based upon the size or density of the antibody-forming cells relative to other cells. Gradients of varying density of solutions of bovine serum albumin can also be used to separate cells according to density. The fraction that is most enriched for desired antibody-forming cells can be determined in a preliminary procedure using the appropriate indicator system in order to establish the antibodyforming cells.

WO 02/16658 PCT/US01/26039 32 The identification and culture of antibody producing cells of interest is followed by enhancement of TERT expression in these cells by the subject methods, thereby avoiding the need for the immortalization/fusing step employed in traditional hybridoma manufacture protocols. In such methods, the first step is immunization of the host animal with an immunogen, typically a polypeptide, where the polypeptide will preferably be in substantially pure form, comprising less than about 1% contaminant. The immunogen may comprise the complete protein, fragments or derivatives thereof. To increase the immune response of the host animal, the protein may be combined with an adjuvant, where suitable adjuvants include alum, dextran sulfate, large polymeric anions, oil water emulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and the like. The protein may also be conjugated to synthetic carrier proteins or synthetic antigens. A variety of hosts may be immunized to produce the subject antibodies. Such hosts include rabbits, guinea pigs, rodents mice, rats), sheep, goats, and the like. The protein is administered to the host, usually intradermally, with an initial dosage followed by one or more, usually at least two, additional booster dosages. Following immunization, generally, the spleen and/or lymph nodes of an immunized host animal provide a source of plasma cells. The plasma cells are treated according to the subject invention to enhance TERT expression and thereby, increase the proliferative capacity and/or delay senescence to produce "pseudo" immortalized cells. Culture supernatant from individual cells is then screened using standard techniques to identify those producing antibodies with the desired specificity. Suitable animals for production of monoclonal antibodies to a human protein include mouse, rat, hamster, etc. To raise antibodies against the mouse protein, the animal will generally be a hamster, guinea pig, rabbit, etc. The antibody may be purified from the cell supernatants or ascites fluid by conventional techniques, e.g. affinity chromatography using RFLAT-1 protein bound to an insoluble support, protein A sepharose, etc.

In an analogous fashion, the subject methods are employed to enhance TERT expression in non-human animals, non-human animals employed in laboratory research. Using the subject methods with such animals can provide a number of advantages, including extending the lifetime of difficult WO 02/16658 PCT/US01/26039 33 and/or expensive to produce transgenic animals. As with the above described cells and cultures thereof, the expression of TERT in the target animals may be enhanced using a number of different protocols, including the administration of an agent that inhibits the TERT expression repression of the target system. The subject methods may be used with a number of different types of animals, where animals of particular interest include mammals, e.g., rodents such as mice and rats, cats, dogs, sheep, rabbits, pigs, cows, horses, and non-human primates, e.g. monkeys, baboons, etc.

SCREENING ASSAYS Also provided by the subject invention are screening protocols and assays for identifying agents that modulate, inhibit or enhance, Myc Repeat region repression of TERT transcription. The screening methods include assays that provide for qualitative/quantitative measurements of TERT promoter controlled expression, of a coding sequence for a marker or reporter gene, in the presence of a particular candidate therapeutic agent.

Assays of interest include assays that measure the TERT promoter controlled expression of a reporter gene coding sequence, luciferase, SEAP, etc.) in the presence and absence of a candidate inhibitor agent, the expression of the reporter gene in the presence or absence of a candidate agent. The screening method may be an in vitro or in vivo format, where both formats are readily developed by those of skill in the art. Whether the format is in vivo or in vitro, an expression system, a plasmid, that includes a Myc Repeat region and a reporter coding sequence all operably linked is combined with the candidate agent in an environment in which, in the absence of the candidate agent, expression of the coding sequence is repressed, in the presence of a combination of Myc and Mad that causes TERT promoter repression. The conditions may be set up in vitro by combining the various required components in an aqueous medium, or the assay may be carried out in vivo, in a cell that normally lacks telomerase activity, an cell, etc.

In certain embodiments, the screening assays are screening protocols and assays for identifying agents that modulate a Myc/Mad transcription WO 02/16658 PCT/US01/26039 34 regulatory system, inhibit or enhance, TERT transcription. By Myc/Mad gene transcription regulatory system is meant a regulatory system in which the expression of a certain coding sequence is controlled by Myc and Mad binding to an E-box repeat region of two or more E-boxes, where in many embodiments, the regulatory system is further characterized in that the repressive activity of Mad dominates Myc, such that when Mad binds by itself to an E-box or when both Mad and Myc (or multiple Myc's) bind to separate Eboxes within the same Myc Repeat region, transcription is repressed. The compositions may be naturally occurring or synthetic, where when they are naturally occurring they are present in other than their natural environment, are isolated, recombinant, etc. In these embodiments, the Myc Repeat region may be a component of the screening assay, as described above.

Alternatively, a nucleic acid component that mimics this region may be employed. For example, a nucleic acid that has an E-box repeat region may be employed, where the E-box repeat region may range in length from about to about 10,000 bases, and usually ranges in length from about 50 to about 5,000 bases. In certain embodiments, the length of the subject E-box repeat region is at least about 700 bases, usually at least about 750 bases and more usually at least about 1000 bases, where the length may be as long as 1000 bases, 5000 bases or longer. The subject E-box repeat region is further characterized by containing a plurality of sequence motifs known in the art as E-boxes, CACGTG. In general, the number of E-boxes present in the subject E-box repeat region may range from about 2 to about 500 or more. In certain embodiments of interest, the number of E-boxes found in the subject Ebox repeat region is at least about 10, usually at least about 15 and more usually at least about 25, where the number may be 50, 100 or higher. In many embodiments, the number of E-boxes found in the subject E-box repeat region ranges from about 10 to 150, usually from about 25 to 125 and is often from about 50 to 100. The E-boxes are positioned in the E-box repeat region relatively close to each other, where the separation distance between any two given E-boxes is typically between about 25 to about 150 bases, usually between about 30 and about 130 bases and often between about 40 and about 50 bases.

WO 02/16658 PCT/US01/26039 A variety of different candidate agents may be screened by the above methods. Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.

Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Agents identified in the above screening assays that inhibit repression of TERT transcription find use in the methods described above, in the enhancement of TERT expression. Alternatively, agents identified in the above screening assays that enhance repression find use in applications where inhibition of TERT expression is desired, in the treatment of disease conditions characterized by the presence of unwanted TERT expression, such as cancer and other diseases characterized by the presence of unwanted cellular proliferation, where such methods are described in, for example, U.S.

Patent Nos. 5,645,986; 5,656,638; 5,703,116; 5,760,062; 5,767,278; WO 02/16658 PCT/US01/26039 36 5,770,613; and 5,863,936; the disclosures of which are herein incorporated by reference.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

A region of the TERT genomic DNA labeled the Myc Repeat region was identified in the course of performing Southern Blots on various cell lines.

The most common, and putative natural size of the Myc Repeat region is around 4500 bases. A smaller version measured to be 2500 bases was also identified. The sequence of this 2500 base Myc repeat region shows that within 1500 bases there are 31 E-Boxes. As such, the natural 4500 base Myc Repeat is expected to have approximately 100 E-Boxes and the smaller 2500 base Myc Repeat has approximately 50 E-Boxes.

The 2500 base Myc Repeat was inserted into the plasmid pSSI-53 (Shown in Fig. 1) to test its affect on expression of the telomerase minimal promoter. The 2500 base Myc Repeat was placed into two different sites upstream of the minimal promoter (See Fig. an XHO1 site and a NOT1 site.

The NOT1 site was used because it is upstream of a transcription blocker and, if increased expression of the telomerase promoter using these constructions were observed, it would be known that the increase in expression was not due to promoter activity within the Myc Repeat, but was, in fact, due to activation of the telomerase promoter. On the other hand, increased expression due to the Myc repeat inserted into the XHO1 site could not be distinguished as such.

However, in all cases, NOT1 insertion, XHO1 insertion, and both orientations of the Myc repeat into each site, showed a 5-10 fold decrease in expression.

The above results indicate that the Myc repeat region or a portion thereof, a region of neighboring E boxes, interacts with one or more transacting factors, E-box binding proteins such as Myc and/or Mad or Myc and/or Mad like proteins, to repress Tert expression. The above results WO 02/16658 PCT/US01/26039 37 also indicate that, in certain embodiments, upon dual binding of Myc and Mad to neighboring E-box sites, Mad dominates to result in transcription repression It is evident from the above results and discussion that the subject invention provides important new nucleic acid compositions that find use in a variety of applications, including the establishment of expression systems that exploit Myc/Mad transcription regulatory systems, such as the regulatory mechanism of the TERT gene, and the establishment of screening assays for agents that enhance TERT expression. In addition, the subject invention provides methods of enhancing TERT expression in a cellular or animal host, which methods find use in a variety of applications, including the production of scientific research reagents and therapeutic treatment applications. It is evident from the above results and discussion that the subject invention also provides important new nucleic acid compositions that find use in a variety of applications, including the establishment of expression systems that exploit the regulatory mechanism of the TERT gene and the establishment of screening assays for agents that enhance TERT expression. Accordingly, the subject invention represents significant contribution to the art.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims (17)

1. A method of making a transgenic plant with increased resistance to the effects of an externally imposed stress selected from the group consisting of water deficit, drought, elevated salinity, and prolonged temperatures below 0 C, relative to non- transgenic wild-type plants, said method comprising: introducing into one or more cells of a plant an exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase in the cells to yield transformed cells; regenerating plants from the transformed cells to yield transgenic plants; 00 10 and selecting a transgenic plant with increased resistance to the effects of an Sexternally imposed stress selected from the group consisting of water deficit, drought, C elevated salinity, and prolonged temperatures below 0°C, relative to non-transgenic wild- type plants, thereby producing a transgenic plant with increased resistance to the effects of an externally imposed stress selected from the group consisting of water deficit, drought, elevated salinity, and prolonged temperatures below 0 C, relative to non- transgenic wild-type plants.

2. A method of making a transgenic plant with enhanced ion accumulation in a plant vacuole of the transgenic plant, relative to non-transgenic wild-type plants, said method comprising: introducing into one or more cells of a plant an exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase in the cells to yield transformed cells; regenerating plants from the transformed cells to yield transgenic plants; and selecting a transgenic plant with enhanced ion accumulation in a plant vacuole of the transgenic plant, relative to non-transgenic wild-type plants, thereby producing a transgenic plant with enhanced ion accumulation in a plant vacuole of the transgenic plant, relative to non-transgenic wild-type plants.

3. A method for increasing production of seeds in plants, said method comprising: providing pollen from a transgenic plant, wherein the transgenic plant has been transformed with exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase to create a transgenic plant;(b) fertilizing a plant with the pollen from the transgenic plant; and(c) culturing the fertilized plant until the plant produces mature seeds.

4. The method of Claim 3, wherein the fertilized plant is the transgenic plant or a plant different from the transgenic plant. A627637spcci The method of any one of Claims 1 to 4, wherein the exogenous nucleic Sacid encodes a vacuolar pyrophosphatase.

6. The method of Claim 5, wherein the exogenous nucleic acid comprises a nucleic acid sequence encoding AVPI, or a homologue thereof.

7. The method of Claim 5 or Claim 6, wherein the nucleic acid sequence c encoding the vacuolar pyrophosphatase is operably linked to one or more regulatory elements comprising nucleic acid that results in overexpression of the vacuolar pyrophosphatase.

8. The method of Claim 7, wherein the nucleic acid sequence encoding the S 10 vacuolar pyrophosphatase is operably linked to one or more regulatory elements c comprising a promoter or chimeric promoter selected from the group consisting of tissue O specific promoters, constitutive promoters, inducible promoters and promoters that are i both tissue-specific and inducible.

9. The method of Claim 8, wherein the nucleic acid sequence encoding the vacuolar pyrophosphatase is operably linked to one or more regulatory elements comprising a promoter that promotes expression of the vacuolar pyrophosphatase in pollen. The method of any one of Claims 5 to 9, wherein the nucleic acid sequence encoding the vacuolar pyrophosphatase is operably linked to one or more regulatory elements comprising a double tandem enhancer of a 35S CaMV promoter.

11. The method of any one of claims 5 to 10 wherein the nucleic acid sequence encoding the vacuolar pyrophosphatase is derived from a wild type plant, a transgenic plant, a mutant plant, or is synthetically derived.

12. A method of making a transgenic plant with increased resistance to the effects of an externally imposed stress, enhanced ion accumulation, or increased resistance to the effects of an externally imposed stress and enhanced ion accumulation, substantially as hereinbefore described with reference to paragraphs 61 to 78.

13. A method of increasing production of seeds in plants, substantially as hereinbefore described with reference to paragraphs 79 to 88.

14. A plant with increased resistance to the effects of an externally imposed stress, enhanced ion accumulation, or increased resistance to the effects of an externally imposed stress and enhanced ion accumulation, produced by the method of any one of claims 1, 2 or 5 to 12. The transgenic plant of claim 14, which has at least increased resistance to the effects of an externally imposed stress selected from the group consisting of water deficit, drought, elevated salinity, and prolonged temperatures below 0°C, relative to non- transgenic wild-type plants.

16. The transgenic plant of Claim 15, which has increased resistance to prolonged exposure to temperatures below 00 C for a 24 hour period or longer. A627637speci 26

17. The transgenic plant of claim 14, which has at least enhanced ion O accumulation in a plant vacuole of the transgenic plant, relative to non-transgenic wild- type plants.

18. The transgenic plant of Claim 14, wherein the plant maintains viability after exposure to a water deficit that is sufficient to compromise the viability of wild-type plants.

19. A transgenic seed of the transgenic plant of any one of Claims 14 to 18 comprising the exogenous nucleic acid. A transgenic progeny of the transgenic plant of any one of Claims 14 to S 0 18. 1 21. A transgenic plant with increased resistance to the effects of an externally O imposed stress selected from the group consisting of water deficit, drought, elevated rC salinity and prolonged exposure to temperatures below 0°C, relative to non-transgenic wild-type plants, said transgenic plant comprising one or more transgenic plant cells, wherein the transgenic plant cells include an exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase in the transgenic plant cells.

22. A transgenic plant with enhanced capacity to accumulate ion species in a vacuole of the transgenic plant, relative to non-transgenic wild-type plants, said transgenic plant comprising one or more transgenic plant cells, wherein the transgenic plant cells include an exogenous nucleic acid that results in increased expression of vacuolar pyrophosphatase in the transgenic plant cells. Dated 30 May, 2007 University of Connecticut Whitehead Institute for Biomedical Research Beth Israel Deaconess Medical Center, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON Ab27637speci