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The invention provides Agrobacterium strains capable of producing a cite-specific recombinase capable of effecting site-specific recombination of a first and a second recombination site in Agrobacterium strains, when present therein, comprising a structural DNA sequence encoding said recombinase and a DNA sequence capable of controlling expression in Agrobacterium strains. The invention also provides methods for using the strains to transform plant cells.
1. An Agrobacterium strain comprising a structural DNA sequence encoding a site-specific recombinase that is Cre recombinase and a DNA sequence linked thereto which operationally controls expression of said Cre recombinase, said strain further comprising a first recombination site.
2. An Agrobacterium strain according to claim 1, wherein said recombination site is located on a plasmid.
3. An Agrobacterium strain according to claim 2, wherein said plasmid is a helper plasmid comprising vir-functions.
4. An Agrobacterium strain according to claim 3, wherein said helper plasmid does not contain T-DNA borders.
5. An Agrobacterium strain according to claim 1, wherein said first recombination site is a loxP site.
6. An Agrobacterium strain according to claim 1, wherein said structural DNA sequence encoding Cre is on a plasmid that is unstable in said Agrobacterium strain.
7. An Agrobacterium strain according to claim 6, wherein said structural coding sequence is under the control of the lac promoter from E. coli.
8. A method for producing a site-specific cointegrate comprising introducing into the Agrobacterium strain of claim 1 a DNA molecule comprising a second recombination site compatible with said first recombination site, and causing said strain to express the Cre recombinase so as to promote recombination between said first and second recognition sites whereby to produce the site-specific cointegrate.
9. A method according to claim 8, further comprising the steps of
effecting a reduced production of the site-specific recombinase, and
selecting Agrobacterium strains comprising the site-specific cointegrate.
10. A site-specific cointegrate obtained using the method of claim 9.
11. A site-specific cointegrate obtained using the method of claim 8.
12. A method according to claim 8 wherein said Agrobacterium strain comprises vir-functions and wherein the DNA molecule also comprises a DNA sequence not naturally present in a plant cell, said DNA sequence not naturally present being positioned on the DNA molecule between a left and a right T-DNA border.
13. A method according to claim 8 wherein said Agrobacterium strain comprises vir-functions and wherein the DNA molecule comprising a second recombination site contains DNA not naturally present in a plant cell, said DNA not naturally present being positioned on the DNA molecule with respect to a right T-DNA border so as to allow said vir-functions to mediate co-transfer of said DNA not naturally present with said right T-DNA border.
14. A site-specific cointegrate obtained using the method of claim 13.
15. Agrobacterium strains comprising the site-specific cointegrate of claim 8.
16. A method for producing a site-specific cointegrate in the Agrobacterium strain of claim 1, with said Cre recombinase mediating integration of first and second DNA molecules when the molecules respectively comprise first and second recognition sites, the method comprising:
a) introducing the first and second DNA molecules into the Agrobacterium strain; and
b) causing said strain to express the Cre recombinase so as to promote recombination between said first and second recognition sites whereby to produce a cointegrate of said first and second DNA molecules in said Agrobacterium strain without homologous recombination.
17. A method according to claim 16 wherein said first and second DNA molecules are free of repetitive DNA sequences that can promote homologous recombination.
18. A method according to claim 16 wherein the Agrobacterium strain is a rec- strain.
19. A method according to claim 16 wherein said second DNA molecule comprises DNA not naturally present in a plant cell and at least a right T-DNA border, said Agrobacterium strain comprising vir-functions to mediate transfer of DNA in the cointegrate to the plant cell, said DNA not naturally present being positioned in the second DNA molecule with respect to the right T-DNA border such that transfer to the plant cell of DNA in the cointegrate mediated by said vir-functions includes transfer of said DNA not naturally present.
20. A method according to claim 19 further comprising:
c) contacting cells of a plant with said Agrobacterium strain so as to cause transfer to the plant of said DNA not naturally present; and
d) selecting a plant cell to which said DNA not naturally present has been transferred.
The invention concerns Agrobacterium strains which harbor recombinant DNA and which are capable of site-specific recombination, a method for obtaining said Agrobacterium strains, and methods of use of said Agrobacterium strains.
Agrobacterium is a genus of gram-negative soil bacteria which cause neoplastic cell growth in dicotyledonous plants by means of the natural system they possess for transferring phytohormone-producing DNA to plant cells. The T-(transferred)region is a segment of the large so-called Ti-(tumour inducing)plasmid (200 kb) harbored by all virulent Agrobacterium strains. (Occasionally, the T-region is also referred to as T-DNA.) Mobilization of the T-region to the plant cells is mediated by another region of the Ti-plasmid, which is called the vir-(virulence)region, in a process that resembles bacterial conjugation (see for reviews: Melchers LB and Hooykaas PJJ (1987) In: Miflin BJ (ed) Oxford surveys of plant and cell biology, vol 4. Oxford Univ Press, London/New York, pp 167-220; Zambryski P (1988) Annu. Rev. Genet. 22:1-30; Zambryski P, Tempe J, Schell J (1989) Cell 56:193-201).
This natural gene transfer system can be used to co-transfer DNA not naturally present in the T-region. The T-region is flanked by two 24 bp imperfect direct repeats (T-DNA borders) which are the only cis-essential elements for the transfer process; i.e. the borders must be linked to the DNA to be transferred to the plant cell. An ideal vector system for transferring DNA to plant cells should comprise the DNA to be transferred into the plant cell flanked on both sides by T-DNA borders, although it has been established that the right T-DNA border, preferably in conjunction with a region just outside the T-region and referred to as "overdrive", suffices for the transfer of DNA. Although not essential, the presence of this overdrive increases the efficiency of transfer of the DNA flanking the right border.
The second essential element, the vir region, can be provided in trans, i.e. not physically linked to the T-region. This finding has originated in the development of a binary vector system, in which the T-region, comprising DNA to be co-transferred to the plant cell, is cloned into a broad-host-range plasmid which is capable of replication in Agrobacterium and E. coli, while the vir functions are supplied by a disarmed helper plasmid which does not contain a T-region (Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) Nature 310:115-120; European Patent EP-B 120 516); Bevan M (1984) Nucl. Acids. Res. 22:8711-8721). A binary vector system wherein the T-region is located on the chromosome of the Agrobacterium strain has also been disclosed (EP-B 176 112).
Although a binary vector system is very versatile, broad-host-range plasmids are less suitable as routine cloning vectors; they are large and generally have a low copy number. Moreover, they are usually unstable in Agrobacterium in the absence of selective pressure.
As an alternative to a binary vector system, one can use a so-called cointegrate system. Typically, the vir-functions reside on a Ti-plasmid and the DNA to be co-transferred has to be recombined into this Ti-plasmid. All cloning steps can be done using an E. coli vector, whereafter this vector is introduced into Agrobacterium, for instance by triparental mating or electroporation. Subsequently, homologous recombination between the vector and the acceptor Ti-plasmid has to take place, via a single or double cross-over, employing large regions of homology between the E. coli vector and the acceptor Ti-plasmid. The regions of homology can for instance be engineered into the acceptor Ti-plasmid by replacing the DNA originally comprised between the T-DNA borders on said plasmid with cloning vector sequences, such as pBR322 (Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J. (1983) EMBO J. 2:411-418; Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M, Schell J. (1983) EMBO J 12:2143-2150; Deblaere R, Bytebier B, De Greve H, deboeck F, Schell J, Van Montagu M, Leemans J (1985) Nucl Acids Res 13:4777-4788). It is also possible to employ helper plasmids which are free of T-DNA borders as acceptor plasmids; in this case it will be necessary to provide at least the right T-DNA border operably linked to the DNA to be introduced into a plant cell on the cloning vector. A disadvantage of this system is that homologous recombination, especially the favor double crossing over, is not a very efficient and precise process in Agrobacterium strains, necessitating tedious selection of the cells harboring the desired cointegrates, before they can be used in plant transformation. An additional disadvantage of the resulting cointegrate plasmids resides in the fact that large regions of repetitive sequences occur, which may cause instability.
It is possible to perform the recombination steps in E. coli instead of Agrobacterium, as described in EP-B 120 515, and subsequently transferring the cointegrate plasmid to Agrobacterium for use in plant transformation.
It would be advantageous if recombination can be performed in Agrobacterium, without the need for large regions of homology between the acceptor plasmid and the cloning vector.
Genetic recombination can be broadly divided into two categories: homologous recombination and site-specific recombination. With homologous recombination the actual site of recombination is not predictable in advance; large regions of homology are necessary to achieve recombination at a reasonable frequency, whereas with site-specific recombination the recombination site is precisely known; moreover, no extensive regions of homology are needed (for review: Craig et al., (1988) Annu. Rev. Genet. 22, 77-105).
A Cre-loxP site-specific recombination system of E. coli phage P1 has been disclosed (Sternberg N and Hamilton D (1981) J Mol Biol 150:467-486.); (lox: locus for recombination--P--from phage P1; cre: causing recombination). The Cre recombinase recognizes the 34 bp loxP DNA sequence consisting of two 13 bp inverted repeats separated by a 8 bp non-symmetric sequence, and promotes its recombination with another loxP sequence. When the two loxP sites are present in different circular DNA molecules, the recombination event causes their cointegration, while when the two loxP sites are in the same molecule, depending on their relative orientation (direct or inverted), the DNA lying in between is either excised or inverted (Abremski K, Hoeas R, Sternberg N (1983) Cell 32: 13011-1311; Hoess RH, Wierzbicki A, Abremski K (1986) Nucl Acids Res 14: 2287-2300).
The system has been shown to work in yeasts (Sauer B (1987) Mol Cell biol 7:2087-2096) and mouse cultured cells (Sauer B and Henderson N (1989) Nucl. Acids. Res. 17:147-161.) and recently, it has been used for site-specific recombination in the genome of transgenic tobacco (Odell J, Caimi P, Sauer B, Russel S (1990) Mol Gert Genet 203:3669-378; International Patent Application WO91/09957).
Similar recombination systems, requiring only a single polypeptide enzyme and short specific DNA sequences (recombination sites) have been reviewed by Craig et al, (supra).
It is an object of the invention to provide Agrobacterium strains and vectors for use therein, which are capable of site-specific recombination, allowing for insertion of DNA not naturally present in said Agrobacterium strains.
The invention provides Agrobacterium strains capable of producing a site-specific recombinase capable of effecting site-specific recombination of a first and a second recombination site in Agrobacterium strains, when present therein, comprising a structural DNA sequence encoding said recombinase and a DNA sequence capable of controlling expression in Agrobacterium strains.
The invention also comprises Agrobacterium strains which further contain a first recombination site.
The invention further provides a method for producing a site-specific cointegrate in an Agrobacterium strain, comprising the steps of
introducing into an Agrobacterium strain which contains a first recombination site a DNA molecule harboring a second recombination site compatible with said first recombination site, and
effecting production of the site-specific recombinase in said Agrobacterium strain.
According to a preferred embodiment of the invention the said Agrobacterium strain harbors vir-functions, and said DNA molecule harboring a second recombination site comprises DNA not naturally present in a plant cell positioned to a right T-DNA border, or positioned between a left and a right T-DNA border, so as to allow co-transfer of said DNA with said right T-DNA border.
The invention further comprises site-specific cointegrates and Agrobacterium strains comprising said site-specific cointegrates.
The invention further comprises a method for obtaining a plant cell containing DNA not naturally present therein, comprising the steps of
contacting plant cells with an Agrobacterium strain harboring a site-specific cointegrate comprising virulence functions and DNA not naturally present in a plant cell positioned to a right T-DNA border, or positioned between a left and a right T-DNA border, so as to allow co-transfer of said DNA with said right T-DNA border, and
selecting a plant cell having obtained said DNA not naturally present therein.
The invention further provides plant cells and plants as well as products and extracts obtained using a method according to the invention.
The invention further comprises Agrobacterium strains which comprise two compatible recombination sites which are in the same orientation and on the same molecule, so that upon production of the recombinase in said Agrobacterium strains recombination is effected and the DNA in between the recombination sites is excised from said vector.
In another embodiment of the invention the latter Agrobacterium strains comprise two compatible recombination sites which are in the opposite orientation and on the same molecule, so that upon production of the recombinase in said Agrobacterium strains recombination is effected and the DNA flanked by the recombination sites is inverted in said molecule.
In a preferred embodiment of the invention said structural DNA sequence is acre coding gene sequence or a functional variant or portion thereof, and said DNA recombination site is a loxP locus, or functional variant or portion thereof.
FIG. 1 shows schematically the construction of pRL756, harboring the cre structural coding sequence under the control of the promoter from the lac-operon from E. coli, a tetracyclin resistance gene and a carbenicillin resistance gene on a plasmid which is unstable in Agrobacterium in the absence of selection.
FIG. 2 shows the relevant features of pMOG621 and pMOG579.
FIG. 3 shows schematically the construction of pAL1166, harboring the vir-functions from pTiB6 and in which the T-region (including the T-DNA borders), has been exchanged with a DNA fragment containing a spectinomycin resistance gene and a loxP recombination site, via homologous recombination. This plasmid is useful for obtaining site-specific cointegrates which may subsequently be used for the transfer of DNA to plant cells. Since pAL1166 no longer contains T-DNA borders, a second DNA molecule containing the DNA to be introduced into plant cells should also provide at least a right T-DNA border operably linked thereto.
FIG. 4 schematically shows cointegration of pAL1166 and pRL754 via site-specific recombination of the loxP sites on both plasmids (upper right part); the lower part shows potential cointegration of pRL754 and pRL756 via homologous recombination, due to the presence of regions of homology between these two plasmids. The latter event, if significant at all, can be avoided if the cre plasmid and the plasmid harboring the DNA to be introduced share no homology or as little as possible.
FIG. 5 represents a generalized scheme for obtaining cointegrates which contain a manipulated T-region for transfer to plant cells; the T-DNA comprises a gene of interest, and, if desired, a marker gene allowing for selection or screening of transformed plant cells. chr=Agrobacterium chromosome; M1 marker gene 1 for selection in Agrobacterium; pTi(δT-DNA) a disarmed (deleted T-DNA) Ti-plasmid harboring vir-functions; M2 marker gene 2 for selection in Agrobacterium; M3 marker gene 3 for selection in Agrobacterium; M1r M3r (M2s) phenotype resistance to marker 1 and 3, and sensitivity to marker 2; LB=left border; RB=right border; incP=plasmid of incompatibility group P.
FIG. 6 is a diagram of pMOG23, harboring a chimeric kanamycin resistance gene for selection in plants, comprising promoter region from the nopaline synthase (nos) gene of Agrobacterium tumefaciens, which is capable of controlling gene expression in plant cells, a structural coding sequence encoding the neomycin phosphotransferase, and the terminator region of the nos-gene.
FIG. 7 diagrammatically illustrates the construction of pAL1766, a site-specific cointegrate, harboring the chimeric plant expressible kanamycin resistance gene flanked on both sides by T-DNA borders, and the virulence functions from pAL1166. This plasmid was used to transfer the kanamycin resistance gene to tobacco cells and regenerate transformed tobacco plants.
FIG. 8 represents a--non-exhaustive--overview of the various possibilities that are offered by the present invention. column A, B and C represent Agrobacterium strains having the cre+ phenotype, e.g. by virtue of the presence of the cre recombinase gene on an incP plasmid (not drawn), or on a stable DNA molecule in Agrobacterium, provided that expression can be regulated through a represssor, an inducer or both; Roman figures represent process steps; Column B represents Agrobacterium strains obtained by either one of the process steps I-VI. Step VII indicates three alternatives (a)-(c) for changing the cre+ phenotype into a cre- phenotype (absence of the recombinase); (a) represents adding a repressor to silence otherwise constitutive expression of the recombinase gene; (b) represents the removal of an inducer of an otherwise repressed expression of the recombinase gene; and (c) represents the removal of the recombinase gene by removal of a plasmid that is unstable in said Agrobacterium strains.
FIG. 9 is a autoradiograph of a Southern blot, showing the results obtained with the cointegrate experiments; for more detailed explanation see Example III-B.
The steps that led to the findings of the present invention, as well as a number of the various ways of practicing the invention are worked out below, in more detail.
To establish whether it was possible to obtain Agrobacterium strains capable of site-specific recombination, the site-specific recombinase Cre and the recombination site loxP were tested in Agrobacterium tumefaciens.
A DNA fragment encoding the structural coding sequence of the cre gene was cloned and positioned under the control of the promoter from the lac-operon from E. coli. This promoter is inducible in E. coli but known to be constitutive in the absence of the repressor in Agrobacterium. This cre gene construct was cloned into an incP plasmid, pNJ5000, yielding pRL756, which appeared unstable in Agrobacterium tumefaciens strain MOG101 in the absence of tetracycline or carbenicillin.
As a first substrate molecule for Cre-mediated site-specific recombination a disarmed (without T-region) helper plasmid was constructed from pTiB6. This was done in Agrobacterium tumefaciens strain LBA1010, by exchanging the T-region from pTiB6 with a DNA fragment containing a loxP-site and a spectinomycin marker gene, via homologous recombination (double crossing-over), employing regions of homology outside the T-DNA borders on pTiB6. This plasmid, derived from pTiB6, now harboring the vir-functions for T-DNA transfer to plant cells, a loxP locus, the spectinomycin marker and no T-DNA borders, was called pAL1166. To establish that Cre-mediated site-specific recombination between the loxP site of pAL1166 and a loxP site of a second DNA molecule can indeed take place in Agrobacterium tumefaciens, a second DNA molecule, pRL754 harboring a loxP site and a kanamycin resistance gene under the control of a promoter on a plasmid derived from pIC20H, was introduced into Agrobacterium strain that harbored both pRL756 and pAL1166, and cointegrate formation was checked by selecting for kanamycin resistance. In the absence of the Cre plasmid pRL756, which was removed in the absence of selection for Carbenicillin and Tetracycline, the cointegrates appeared stable.
From these experiments it was concluded that Cre-mediated site-specific recombination between a first and a second loxP site is a very efficient process in Agrobacterium tumefaciens, and that the cointegrates remain stable, which opens new perspectives for the introduction of DNA into Agrobacterium which is not naturally present therein.
To find out whether cointegrates obtained via Cre-mediated site-specific recombination can also be used for plant transformation, the above experiment was repeated in a slightly modified way. Into plasmid pRL754, shown to be cointegrated into pAL1166 in the above experiment, a DNA fragment containing a left T-DNA border, a plant expressible kanamycin resistance gene under the control of the promoter of the nopaline synthase gene of Agrobacterium tumefaciens, and a right T-DNA border. This plasmid was introduced into the Agrobacterium tumefaciens strain harboring the Cre plasmid pRL756 and plasmid pAL1166, and strains harboring Cre-mediated site-specific cointegrates were selected as above.
To assess whether Agrobacterium strains harboring these site-specific cointegrates are capable of co-transferring the plant expressible kanamycin resistance gene located between the T-DNA borders to plant cells, the Km resistant Agrobacterium strains were used for transformation of tobacco plants. It was found that Agrobacterium strains harboring these site-specific cointegrates were equally capable of effective transfer of T-DNA to plant cells as comparable strains harboring the same manipulated T-region on binary vectors.
Transformed tobacco plants are now grown to maturity and allowed to set seed. It is predicted that the stability of the kanamycin resistant phenotype does not differ from conventionally transformed plants, and that the trait is transferred to the progeny.
It will be clear from the experiments above that site-specific recombination makes Agrobacterium a more versatile system, both if used as a plant transformation system and as a host for foreign DNA expression per se.
DNA to be introduced into Agrobacterium, e.g. modified T-regions for plant transformation harboring gene cassettes to be transferred to plant cells, or genes meant to be expressed in Agrobacterium itself, can be cloned suitably in small and sophisticated cloning vectors for any suitable host, said vectors only requiring to comprise a recombination site, which is easily inserted into said vector as a small DNA fragment.
Since this site-specific recombination system is completely independent of conventional homologous recombination, large regions of hemology are not necessary to achieve recombination, and one can even work in an Agrobacterium strain that is deficient for homologous recombination (rec-), thus further reducing the occurrence of unwanted rearrangements which may be due to coincidental regions of hemology.
As is reflected diagrammatically in FIG. 8, site-specific recombination according to the invention is intended to cover both cointegration, excision and inversion.
Cointegration, whereby at least two DNA molecules recombine to form at least one larger DNA molecule, is reflected in steps I, II, and III. Step I indicates the possibility to simultaneously introduce into Agrobacterium at least two DNA molecules capable of site-specific recombination. In this particular situation at least one of both DNA molecules should contain an origin of replication that is functional in Agrobacterium, should it be desired that the cointegrate is stably maintained therein; steps II and III indicate the formation of a cointegrate where at least one DNA molecule is resident and another DNA molecule is newly introduced, whereby the resident DNA molecule in step II is a chromosome of Agrobacterium, and in step III the resident molecule is a plasmid. Obviously, this is not indicated, it would be possible to introduce DNA molecules into cre- Agrobacterium strains, and furnish the Cre-recombinase for instance by introducing a plasmid containing the gene, simultaneously, or after the introduction of the said DNA molecules.
Step VII is a preferred step for stabilizing a formed site-specific cointegrate obtained after steps such as I-III; (a) refers to adding to Agrobacterium a repressor for the cre-gene, alternatively, or additionally (b) the inducer of normally repressed gene can be removed, or (c) the gene encoding the Cre-recombinase can removed in its entirety by allowing an unstable plasmid to become lost from the Agrobacterium strains.
Steps IV-VI indicate the possibility to excise or invert fragments of DNA between two recombination sites.
According to a preferred embodiment of the invention the Agrobacterium strains according to the invention are used as a transfer system of DNA to plant cells.
Preferred DNA to be transferred to plant cells are genes encoding protein, and a expression controlling DNA sequence such that upon expression of the gene the protein is produced in a plant or plant cell at the desired stage and at the desired site in the plant. The gene of interest may also comprise genes which can be expressed in the form of an RNA sequence which does not encode protein, such as antisense genes, ribozyme genes and the like. The gene of interest not necessarily needs to be capable of being transcribed; it may as well be a recognition sequence that can be recognized by proteins, e.g. a recombinase, a nuclease and the like, or by man, serving as a genetic label.
More specific examples of the use of a gene of interest include but are not limited to those involved with fungal resistance (EP-A 392 225, International Patent Application WO90/07001; EP-A 440 304), insect resistance (EP-A 193 259), nematode resistance (EP-A 352 052), virus resistance (EP-A 223 452), altered carbohydrate composition (WO90/12876; EP-A 438 904), altered oil composition (EP-A 225 377), seed storage proteins with altered amino acid composition (EP-A), male sterility (EP-A 329 308), modified flower color (EP-A 335 451), delayed fruit ripening (WO91/01375), salt resistance (WO91/06651), herbicide resistance (EP-A 218 571; EP-A 369 637), production of pharmaceutical products (EP-A 436 003) and the like.
Suitable DNA sequences for control of expression of genes of interest, or marker genes, such as promoters, enhancers, non-transcribed leaders and the like, may be derived from any gene that is expressed in a plant cell, including plant genes (EP-122 791), genes located on wild-type T-DNA of Agrobacterium (EP-A 126 546), plant virus genes (EP-B 131 623), including functional portions, hybrids, or synthetic copies thereof.
To select or screen for transformed cells, it is preferred to include marker genes linked to the gene of interest in the T-region that is to be transferred to the plant cell. The choice of a suitable marker gene in plant transformation is well within the scope of the average skilled worker; some examples of routinely used marker genes are the neomycin phosphotransferase genes conferring resistance to kanamycin (EP-B 131 623), the Glutathion-S-transferase gene from rat liver conferring resistance to glutathion derived herbicides (EP-A 256 223), glutamin synthetase confering upon overexpression resistance to glutamine synthetase inhibitors such as phosphinotricin (WO87/05327), the acetyl transferase gene from Streptomyces viridochromogenes confering resistance to the selective agent phosphinotricin (EP-A 275 957), the gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine, the bar gene conferring resistance against Bialaphos (e.g. WO91/02071) and the like. The actual choice of the marker is not crucial as long as it is functional (i.e. selective) in combination with the plant cells of choice.
The marker gene and the gene of interest do not have to be linked, since co-transformation of unlinked genes (U.S. Pat. No. 4,399,216) is also an efficient proces in plant transformation.
Transformed cells obtained and selected according to the invention may be used as such, for instance for the production of a pharmaceutical compound in cell suspension cultures, or used to generate a whole new plant.
The choice of plant material for obtaining transformed plant cells and/or generation of whole new transformed plants is not critical to this invention as long as it is amenable to T-DNA transfer by Agrobacterium strains.
Especially preferred plant material, especially for dicotyledonous crops are leaf-discs which can be readily transformed and have good regenerative capability (Horsch R. B. et al., (1985) Science 227, 1229-1231).
Monocotyledonous plants are amenable to DNA transfer by Agrobacterium strains (EP-B 159 418), including commercially important crops such as corn (Gould J, Michael D, Hasegawa O, Ulian EC, Peterson G, Smith RH, (1991) Plant. Physiol. 95, 426-434).
Recent reports suggest that Agrobacterium mediated DNA transfer may be combined with tissues that have been wounded by microprojectile bombardment (Bidney D, Scelonge C, Martich J, Burrus M, Sims L, Huffman G, (1992), Plant Mol Biol 18, 301-313).
It should be clear that the Agrobacterium strains according to the invention can be used for plant transformation just as `conventional` Agrobacterium strains using the same techniques as employed with conventional Agrobacterium strains. It is therefore clear that alternative ways of using Agrobacterium for plant transformation, not explicitly mentioned here, do not depart from the scope of the invention.
All references cited in this specification are indicative of the level of skill in the arts to which the invention pertains. All publications, whether patents or otherwise, referred to previously or later in this specification are herein incorporated by reference as if each of them was individually incorporated by reference.
The Examples given below are just given for purposes of enablement and do not intend in any way to limit the scope of the invention.
E. coli MH1 (Goddard JM, Caput D, Williams SR, Martin Jr DW (1983) Proc Natl Acad Sci U.S.A. 80:4281-4285) and JM101 (Yanisch-Perron C, Vieira J, Messing J (1985) Gene 33:103-119) were used for cloning. A. tumefaciens strains were: LBA1010 (C58, pTiB6, Rif) (Koekman et al., (1982) Plasmid 7, 119-132), MOG1010 (derived from LBA1010 by substitution of the T-DNA by a spectinomycin marker) and UIA143 (C58 recA mutant, pTi-cured, Ery) (Farrand SK, O'Morchoe SP, McCutchan J (1989) J Bacteriol 171: 5314-5321). Plasmids used in this work were: pUC19 (Yanisch-Perron et al, 1985, supra), pIC20H (Marsh JL, Erfle M, Wykes EJ (1984) Gene 32:481-485; FIG. 3), pHP45ΩKm (pBR322 derivative containing a kanamycin gene) (Fellay R, Frey J, Krisch H (1987) Gene 52:147-154), pMOG621 (pBR322 derivative containing two pTiB6 T-DNA flanking fragments), pMOG579 (pMOG621 with a spectinomycin cassette separating the two pTiB6 fragments; FIG. 2), pNJ5000 (RP4 unstable derivative, Tc; FIG. 1) (Grinter NJ (1983) Gene 21:133-143), pBIN19 (incP binary vector, Km) (Bevan, 1984, supra), and pRK2013 (Ditta G, Stanfield S, Corbin D, Helinski D (1980) Proc Natl Acad Sci U.S.A. 77:7347-7351). P1 phage used was Plclr100Cm.
E. coli cells were grown in LC medium (Hooykaas PJJ, Klapwijk PM, Nuti MP, Schilperoort RA, Rorsch A (1977) J Gen Microbiol. 98:477-484) at 37� C. (P1 containment cells). Antibiotics were added to the following concentrations: carbenicillin, 100 μg/ml; kanamycin, 25 μg/ml; spectinomycin, 50 μg/ml; tetracycline, 5 μg/ml; cloramphenicol, 50 μg/ml. Agrobacterium strains were grown in LC medium (Hooykaas et al, 1977, supra) at 29� C. Antibiotic concentrations used were; carbenicillin, 75 μg/ml; kanamycin, 100 μg/ml; spectinomycin, 250 μg/ml; tetracycline, 2 μg/ml; rifampicin, 20 μg/ml; erythromycin, 100 μg/ml.
DNA isolation from E. coli, restriction, ligation, transformation, nick-translation of probes, and Southern blot to nitrocellulose filters were performed according to standard techniques (Sambrook J. Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Plasmid DNA was isolated from Agrobacterium by a modification of the alkaline lysis procedure of Birnboim and Doly (Birnboim HC, Doly J (1979) Nucl. Acids Res. 7:1513-1523), in which an extra denaturing step was included by the addition of a NaOH-phenol solution (2�0.2N NaOH, 1� Tris-saturated phenol) to the alkaline lysate (1/10 vol.). Hybridization was carried out at 42� C. in 50% formamide, 5� SSC, and the hybridized filters were washed at 42� C. in 2� SSC, 0.1% SDS.
Plasmids were routinely introduced into the Agrobacterium strains by electroporation, as described (Mozo T, Hooykaas PJJ (1991) Plant Mol. Biol. 16:917-918). Triparental matings according to Ditta et al. (supra) were used when a double cross-over was intended.
PCR was performed in a Biozym-PREM processor. The DNA isolated form 0.5 ml of an overnight culture of P1 containing E. coli cells was EcoRI digested (the cre gene has no EcoRI sites (Sternberg N, Sauer B, Hoess R, Abremski K (1986) J. Mol. Biol 187:197-212), and used for PCR in a final volume of 100 μl. The reaction mixture contained: 50 mM KCl, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 100 μg/ml BSA, 50 μM of each nucleotide (Pharmacia), 0.45 μM of each primer and 5 U of Taq polymerase (Promega). Reactions were overlaid with 75 μl of mineral oil. The mixture war heated at 93� C. for 2 min to denature the DNA, cooled at 55� C. for 1 min to anneal the primers, and heated again to 72� C. for 1 min to initiate the amplification. The actual amplification took place during 20 cycles of 93� C.--1 min, 55� C.--1 min, 72� C.--1 min, followed by a final elongation step of 5 min at 72� C. The mixture was finally extracted twice with chloroform to remove the oil. The primers were designed to obtain a final 5' SphI -3' BcORI cre gene, with no native promoter (Sternberg et al., supra). The sequence of the upstream primer was 5'-GGGCATGCGGAGTGTTAAATGTCC-3' (SEQIDNO: 1), and the downstream primer was 5'-GGGAATTCATGGCTAATCGCCATC-3' (SEQIDNO: 2) (start and stop codons are underlined).
A 41 bp synthetic BamHI-EcoRI loxP sequence was constructed from the oligonucleotides: P1, 5'-GGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATG-3'(SEQIDNO: 3), and P2, 5'-GAATTCATAACTTCGTATAGCATACATTATACGAAGTTATG-3' (SEQIDNO: 4).
Obtention of Agrobacterium Strain MOG101
A helper plasmid conferring the Agrobacterium tumefaciens virulence functions derived from the octopine Ti-plasmid pTiB6 was constructed, MOG101. MOG101 is a Agrobacterium tumefaciens strain carrying a non-oncogenic Ti-plasmid from which the entire T-region was substituted by a bacterial Spectinomycin resistance marker from transposon Tn 1831 (Hooykaas et al., 1980 Plasmid 4, 64-75).
The Ti-plasmid pTiB6 contains two adjacent T-regions, TL (T-left) and TR (T-right). To obtain a derivative lacking the TL- and TR-regions, we constructed intermediate vector pMOG579. Plasmid pMOG621 is a pBR322 derivative, which contains the 2 Ti-plasmid fragments that are located to the left and right, outside the T-regions (FIG. 2). In pMOG579 the 2 fragments (shown in dark) were separated by a 2.5 kb BamHI-HindIII fragment from transposon Tn1831 (Hooykaas et al., 1980 Plasmid 4, 64-75) carrying the spectinomycin resistance marker (FIG. 2). The plasmid was introduced into Agrobacterium tumefaciens strain LBA1010 [C58-C9 (pTiB6)=a cured C58 strain in which pTiB6 was introduced (Koekman et al. (1982), Plasmid 7, 119-132) by triparental mating from E. coli, using HB101 8pRK2013) as a helper. Transconjugants were selected for resistance to Rifampicin (20 mg/l) and spectinomycin (250 mg/l). A double recombination between pMOG579 and pTiB6 resulted in loss of carbenicillin resistance (the pBR322 marker) and deletion of the entire T-region. Of 5000 spectinomycin resistant transconjugants, replica plated onto carbenicillin (100 mg/l) 2 were found sensitive. Southern analysis showed that a double crossing over event had deleted the entire T-region (not shown). The resulting strain was called MOG101. This strain and its construction is analogous to strain GV2260 (Deblaere et al. 1985, Nucl. Acid Res. 13, 4777-4788).
Construction of pRL756 Harboring the Cre Gene on a Plasmid Unstable in Agrobacterium
The PCR fragment containing the cre gene was digested with SphI and EcoRI and cloned into the polylinker site of plasmid pUC19 (FIG. 1). The resulting construct contains the cre-gene under the control of the lac promoter. The resulting pUC19-cre plasmid was subsequently cloned into the unique EcoRI site of the incP plasmid pNJ5000. The Cb marker of the pUC19 plasmid, allowing positive selection for the recombinant clones, made straightforward an otherwise highly difficult cloning step, given the large size (44 kb) and low copy number of the plasmid pNJ5000. The stability of the final cre-construct (pRL756) was checked in the Agrobacterium strain MOG1010. In these experiments, between 50% and 70% of the population lost the plasmid after 24 hours of growth in liquid medium in the absence of both carbenicillin and tetracycline, while pRL756 was stably maintained in the Agrobacterium cells when both antibiotics were included in the culture medium (data not shown).
Construction of a pTi-loxP Disarmed Helper
An EcoRI loxP-Spc cassette was constructed in two steps in plasmid pIC20H (details of the cloning procedures are shown in FIG. 3). This cassette was subsequently cloned into the EcoRI site of plasmid pMOG621. This plasmid contains two fragments from pTiB6 which flank the T-DNA outside the left and right borders in the original pTiB6 and can therefore, be used as intermediate for T-DNA elimination or substitution by double homologous recombination at these two pTi fragments. The resulting plasmid pMOG622 was introduced by triparental mating into Agrobacterium strain LBA1010, and double recombinants in which the T-DNA had been exchanged by the loxP-Spc cassette were selected by resistance to spectinomycin and sensitivity to carbenicillin (indicating loss of the pBR322 vector part of pMOG622). The presence of the loxP-Spc cassette in the resulting pLBA1010 (δT-DNA)::loxP-Spc disarmed helper was checked by Southern blot (FIG. 9). This plasmid was called pAL1166 (see FIG. 3).
Cre-mediated Cointegration Experiments
In order to determine whether the two plasmids described above can actually mediate loxP-site specific recombination, a third plasmid was constructed by cloning a kanamycin resistance gene into the EcoRI site of plasmid pRL753 (FIG. 3). The resulting plasmid pRL754 was used to check cointegrate formation after electroporation into Agrobacterium strains containing the loxP system by selection for Kmr colonies suggesting that site-specific recombination has taken place. The results are discussed more extensively in Example III-B.
Control Experiments and Evidence for Site-specific Cre Mediated Cointegration
A number of experiments were carried out to provide evidence that Cre-loxP mediated site-specific recombination accounted for cointegrate formation.
Besides the strain containing both the pTi-loxP disarmed helper pAL1166 and the Cre-plasmid pRL756, strains containing only pAL1166 or a normal pTi helper plus pRL756 were used as control. In addition, a strain in which pRL757 had been integrated into MOG101 by single recombination at the Spc marker gene was included in the experiments in order to compare the efficiency of the loxP-Cre mediated and the normal homologous recombination processes. In a parallel experiment and as a way to assess that all the Agrobacterium strains used had similar levels of competence for DNA uptake, the same strains were also electroporated with the Kmr plasmid pBIN19. The results of these experiments are presented in Table 1A. As can be seen from the table, the strains used did not differ significantly in their competence for DNA uptake of the control plasmid pBIN19. However, none or a just a single Kmr transformant was obtained when pRL754 was electroporated into the strains that did not contain any of the loxP-Cre elements or only contain the loxP site as homologous recombination between the common pIC20H-loxP sequences was apparent when pRL754 was introduced into the strain containing pRL757 integrated into the pTi plasmid. A similar frequency of homologous recombination was observed for the strain containing pRL756 and no loxP site. In this case, the incoming plasmid and the resident pRL756 have in common the pUC replication and Cb regions (FIG. 4). The homology between these two plasmids posed an important problem to assess loxP-Cre site-specific recombination in the strain containing both the pAL1166 helper and pRL756. Although a slightly higher transformation frequency was obtained by electroporation of pRL754 into that strain, this increase was not enough to be attributed to the addition of loxP-Cre specific recombination events to the already shown highly efficient pUC-Cb mediated homologous recombination, considering that this strain also resulted in more Kmr transformants when electroporated with pBIN19. An indirect approach was followed in order to check whether loxP-Cre site-specific recombination had actually occurred. The Kmr transformants obtained by electroporation of pRL754 into the strains containing pRL756 and either the normal helper pMOG101 or the pTi-loxP helper pAL1166 were separately pooled, grown overnight in liquid cultures with and without kanamycin, and plated in media without antibiotics, with kanamycin and with tetracycline to assay for the stability of the markers (Table 2). These experiments showed that, when growing in antibiotic-free medium, Km and Tc were lost at the same rate (12%) in the case of the cells which did not include the loxP site, thus indicating that Km is linked to Tc in the unstable pRL756 plasmid. In contrast, in the case of the transformants obtained with the pAL1166 helper containing strain, Tc was lost in a 91% of the cells, while the rate for the Km loss was only of 38%. In agreement with that, when this second group of transformants was grown in Km, only 13,5% of the cells retained the Tc marker. These results, showing that there is no linkage between Km and tc markers in the Kmr transformants obtained by electroporation of pRL753, demonstrate that the incoming plasmid is not cointegrated with pRL756 and suggest that the Km resistance is due to integration of pRL754 into pAL1166 by loxP-Cre mediated recombination.
The cointegration between the incoming plasmid and pRL756 may have also occurred, but since this would give rise to different loxP containing plasmids in the same cell, the situation would not be stable, unless the Cre plasmid pRL756 or a putative pRL756::pRL754 cointegrate plasmid would have been lost. This instability is also suggested by the higher rate of Kmr loss from this group of transformants when growing in antibiotic-free medium (38% vs. 12% in the loxP absent transformants).
In order to more definitively assess the loxP-Cre site-specific recombination in our system, the above described electroporation experiments were repeated in a RecA- background (Table 1B), in which no homologous recombination events should occur. As expected, the transformation frequency in the case of the strain containing pRL757 integrated into the pTi was now reduced to the levels obtained when no homologous sequences or only the small loxP site were involved (this levels can be assimilated to the frequency of spontaneous mutations). However, despite the RecA-, the transformation frequencies in both strains containing pRL756 was again similar, independent of the presence of the loxP site. This unexpected result indicates that the Cre protein may mediate recombination at sites different from the loxP sequence. In an attempt to discriminate between these two types of Cre-mediated recombination, the Kmr transformants obtained with both the loxP-present and absent strains were directly checked, without further culturing, for the presence of the Km and Tc resistance markers. While all MOG101(pRL756) transformants were Kmr Tcr, 50% of the pAL1166 transformants were Kmr Tcs, thus indicating that pRL756 had been lost shortly after the loxP-Cre mediated cointegration of pRL754 into the pAL1166. The remaining 50% Kmr Tcr transformants would likely include only pRL754::pRL756 cointegrates because of the stability problems that the coexistence of different loxP containing plasmids in the same cell in the presence of the cre protein may cause, as discussed. The recombination events were checked by Southern blot analysis of four representatives of each type (Tcr and Tcs) of transformants (FIG. 4). The plasmid DNA isolated from those transformants was HindIII digested and hybridized with labelled pRL754. In the case of all Tcr transformants checked, three bands corresponding with the HindIII fragments of a putative pRL757::pRL754 cointegrate (FIG. 8) were observed, together with the HindIII pAL1166 fragment which contains the loxP sequence (FIG. 3). The stronger hybridization signal in the upper band corresponding to the 44 kb pRL756 is likely due to the presence of non-cointegrated copies of this plasmid, since incP plasmids can exist in several copies per cell and the cointegration of the Km-containing incoming plasmid in only one of them should be enough to confer Km resistance. In the Tcs transformants this upper band was absent, as like the pAL1166 fragment containing the loxP site. Three bands were observed in this case corresponding to the expected HindIII fragments of a pAL1166::pRL754 cointegrate obtained by recombination at the loxP site (FIG. 4). Again there is a difference in the relative intensity of the hybridizing bands which can be attributed to the integration of several copies of the incoming plasmid into the pTi-loxP helper plasmid (pAL1166).
These results clearly show that loxP-Cre mediated site-specific cointegrate formation works efficiently in the Agrobacterium cells and is a useful alternative for obtaining cointegrates using homologous recombination.
Construction of a pTi::T-DNA Cointegrate
Having established the process of loxP-Cre mediated cointegrate formation in Agrobacterium, we carried out experiments to introduce a modified T-region into the disarmed loxP-helper plasmid pAL1166. To this aim we subcloned the T-region fragment as contained on the binary vector pMOG23 (in E. coli K-12 strain DH5α, deposited at the Centraal Bureau voor Schimmelcultures on Jan. 29, 1990 under accession number CBS 102.90). As shown in FIG. 6, the entire T-region of pMOG23, carrying a chimeric NPTII gene between its left and right borders, is contained on a BglII-restriction fragment. This fragment was subcloned into plasmid pRL754 (Example III-A3) linearized with the enzyme BamHI.
The resulting plasmid pMOG623 was integrated into pAL1166 as shown in FIG. 7. The Agrobacterium strain carrying pAL1166 and pRL756 (Example II) was electroporated with plasmid pMOG623 selecting for kanamycin resistance. This was followed for screening for loss of TcR, indicative for loss of pRL756. This process resulted in the formation of of a pAL1166-pMOG623 cointegrate, called pAL1766. Its structure is shown in FIG. 7.
The Agrobacterium strain pAL1766 was used for transformation of tobacco plants, next to Agrobacterium strain MOG101 carrying pMOG23. This was done to compare the transformation capability of strains constructed via loxP-Cre mediated cointegrate formation with strains carrying binary vectors.
Transformation of tobacco (Nicotiana tabacum cv. Petit Havana SR1) was done via cocultivation of 50 leaf discs according to the procedure of Horsch et al. (Horsch et al., (1985), Science 227, 1229-1231). In experiments with both strains, the leaf discs were cocultivated with bacteria grown to OD550 of 0.5 during 48 hrs, and transferred to selective media (100 mgs/l kanamycin). After 4-6 weeks shoots were obtained and assayed for proper rooting on kanamycin (100 mgs/l), transferred to soil and allowed to selfpollinate and set seed. Seeds from individual transformants were pooled and germinated on kanamycin (200 mgs/l). From these experiments out of 50 leaf discs a total of 25-30 transgenic plants was recovered. No differences were observed in numbers or appearance of plants obtained via either vector system.
Deposit of Microorganisms
Agrobacterium tumefaciens strain LBA1166 harboring pAL1166 has been deposited at the NCC, Julianalaan 67, Delft, The Netherlands, on Feb. 26, 1992, under deposit number cbs
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 4(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24(B) TYPE: Nucleic Acid(C) STRANDEDNESS: Single(D) TOPOLOGY: Linear(ii) MOLECULE TYPE: cDNA(iii) HYPOTHETICAL: Yes(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GGGCATGCGGAGTGTTAAATGTCC24(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24(B) TYPE: Nucleic Acid(C) STRANDEDNESS: Single(D) TOPOLOGY: Linear(ii) MOLECULE TYPE: cDNA(iii) HYPOTHETICAL: Yes(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GGGAATTCATGGCTAATCGCCATC24(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 41(B) TYPE: Nucleic Acid(C) STRANDEDNESS: Single(D) TOPOLOGY: Linear(ii) MOLECULE TYPE: cDNA(iii) HYPOTHETICAL: Yes(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATG41(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 41(B) TYPE: Nucleic Acid(C) STRANDEDNESS: Single(D) TOPOLOGY: Linear(ii) MOLECULE TYPE: cDNA(iii) HYPOTHETICAL: Yes(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GAATTCATAACTTCGTATAGCATACATTATACGAAGTTATG41__________________________________________________________________________
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Swan Veterinary Practice is one of the few vets still offering drop in consultations where you can have your pet looked at without an appointment booked in advanced through our Holburn Street Practice. We have been providing veterinary services across the North East of Scotland for over twenty years and pride ourselves on our welcoming atmosphere and our friendly and approachable staff. We are a small animal clinic, providing a variety of services and treatments for your pets.
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The Best Dresses at the 2012 SAG Awards
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Celebutante Paris Hilton rocked out at TAO Nightclub at The Lift in these uber-cool black furry earmuffs.


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American Cancer Society http://www.cancer.org
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Spinal tumors. American Association of Neurological Surgeons website. Available at: http://www.aans.org/Patient%20Information/Conditions%20and%20Treatments/Spinal%20Tumors.aspx. Updated February 2012. Accessed June 4, 2013.
Spinal cord tumor. University of California San Francisco Medical Center website. Available at: http://www.ucsfhealth.org/conditions/spinal%5Fcord%5Ftumor/. Accessed June 4, 2013.
Spinal tumors. EBSCO DynaMed website. Available at: http://www.ebscohost.com/dynamed. Updated March 20, 2013. Accessed June 4, 2013.
Spinal tumors. University of Texas MD Anderson Cancer Center website. Available at: http://www.mdanderson.org/patient-and-cancer-information/cancer-information/cancer-types/spinal-tumors/index.html. Accessed June 4, 2013.


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Baldwinsville The Baldwinsville Kiwanis Club will host the 43rd annual Baldwinsville Kiwanis Turkey Trot on Thanksgiving morning, Nov. 24, at Charles W. Baker High School, 29 East Oneida St., Baldwinsville.
The annual Turkey Trot features a half-mile Kids Fun Run in addition to 5K and 10K races. The kids run starts at 8:30 a.m., the 5K at 9:15 a.m. and the 10K at 10 a.m. Proceeds from the Baldwinsville Kiwanis Turkey Trot will benefit Ronald McDonald Charities of Central New York, Kamp Kiwanis and other charities that help meet the needs of children.
In 2009, the Baldwinsville Kiwanis Club pledged $25,000 to Ronald McDonald House Charities of CNY to help build a new, larger handicap-accessible Ronald McDonald House. The event also provides funding to send local children to Kamp Kiwanis, a coed camp for children ages 8 to 14 that is aimed at providing fun, physical exercise and adventure.
Registration is $25 on the day of the race. Kids 12 and under are free. To register online, visit baldwinsvillekiwanis.com. First 1,800 pre-registered entries will receive a free T-shirt.
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Belmont Stakes Raceday
Belmont Stakes Raceday
Belmont Stakes Raceday
By David Harrison
It could be a historic day at Belmont Park on Saturday, as Big Brown tries to become only the 12th horse ever and the first in 30 years to win the elusive Triple Crown in the Belmont Stakes.
Either due to his own dominance or to the questionable strength of the field around him, Big Brown is a huge favorite to win the Belmont at 2/5. It looked like Big Brown's run at Affirmed - the last Triple Crown winner - might be in jeopardy a few weeks ago after a crack was discovered in his left front foot. However, the crack was found to be very minor and Big Brown went through his regular training regimen without any problems leading up to the Belmont. For added safety a synthetic patch was applied to the hoof on Friday which should all but erase any doubts regarding Big Brown's health.
Judging by the rest of the odds for the 140th running of the Belmont, the only horse the oddsmakers think has a real chance to upset Big Brown is Casino Drive, listed at 7/2. In his very short racing career Casino Drive is undefeated in two races, both of which were impressive victories. In his maiden, which took place in Japan, Casino Drive utterly destroyed the rest of the field, winning by 11 ½ lengths. In his first race on American soil, Casino Drive won the Peter Pan Stakes by 5 ¾ lengths.
Following Big Brown and Casino Drive on the Belmont odds is a former challenger of Big Brown, Denis of Cork at 12/1. Denis of Cork last raced in the Kentucky Derby and finished third behind Big Brown and the late Eight Belles. Also returning from the Derby field to take another shot at Big Brown is Tale of Ekati at 20/1 and Anak Nakal at 30/1. Tale of Ekati finished fourth in the Derby, while Anak Nakal was seventh.
There will also be two horses from the Preakness Stakes field running in the Belmont. They include Macho Again, a 20/1 Belmont underdog, and Icabad Crane, also listed at 20/1. Macho Again finished as runner-up to Big Brown, while Icabad Crane followed him in third place.
Odds to win Belmont Stakes
Big Brown 2/5
Casino Drive 7/2
Denis of Cork 12/1
Icabad Crane 20/1
Macho Again 20/1
Tale of Ekati 20/1
Anak Nakal 30/1
Da' Tara 30/1
Ready's Echo 30/1
Guadalcanal 50/1
Re: Belmont Stakes Raceday
Belmont Analysis
What a great day for racing. We have a ligitimate shot at the first triple crown winner in 30 years. From a betting standpoint there is not much value and Big Brown is a legitimate odds-on favorite for the final leg. It is hard to go past and quite frankly, as a fan of racing, I dont want to be rooting against the best shot at the honor in 30 years. Here is a quick synoposis of the field from my perspective:
Big Brown: Class of the field. Hoof little worry should handle a pretty weak group of three year olds
Guadalcanal: A maiden against this Big boy; NOPE Macho Again: Trouble in Preakness, still way back.
Denis of Cork: Skipped middle leg after needing the Derby; where he didn't come into the race on best of terms
Casino Drive: Japanese hope is the wildcard but hard to come back here after Peter Pan
Da'Tara: Zito says he'll go to the front, can you spell DOOM
Tale of Ekati: Won slow Wood here, dont like Tagg and a minor share is all I see from this guy
Anak Nakal: Zito's other horse is looking to benefit from fast pace. May be better later in the year
Ready's Echo: 3rd in Peter Pan, another pace factor. Pletcher owns track but dont think this guy is good enough
Icabad Crane: Had some trouble at key point in the Preakness. May be second best here and might have a big one in him; Second Choice
Look, the race is Big Brown's to lose. The pace could be crazy fast and if Kent gets sucked up too far or taken out of his game something strange could happen.
Big Brown is yet to face adversity and have to chase into the stretch. He looks very tough to beat. Icabad Crane had some trouble in last and may have been closer at the end. Because he may be some value and i think he has the best shot at an upset, I will place a few dollars on Icabad Crane to satisfy my 'value' side. Enjoy the races
Re: Belmont Stakes Raceday
Big Brown figures to draw plenty of action for Belmont Stakes
There are several proposition bets for the horse favored to become the first Triple Crown winner in 30 years.
Ten horses are scheduled to compete in today's Belmont Stakes in New York but most attention will be placed on Triple Crown contender Big Brown.
After winning the Kentucky Derby and the Preakness Stakes, Big Brown is listed as a solid 2-5 favorite to win the Belmont and become the first horse in 30 years to capture the Triple Crown.
For gamblers interested in bigger payouts involving Big Brown in the Belmont, there are numerous proposition bets available at Bodoglife.com. Here are a few of them:
What will Big Brown's margin of victory be? 1-5 lengths (1-4), 6-10 lengths (3-1), or more than 10 lengths (11-1); Which position will Big Brown be at the official quarter mile mark? First or second place (even), third to fifth place (5-7) or sixth place or higher (14-1).
Will Big Brown win going wire to wire? Yes (+450) or No (-750); Will Big Brown take the lead and lose it? Yes (+175) or No (-250); Will Big Brown tie or beat Secretariat's record of a 31-length win in 1973? Yes (+25,000) or No (-50,000).
There are also plenty of prop bets involving other horses in the race, but those involving Casino Drive were removed after it became known he had a hoof injury and was held out of a scheduled workout Friday.
Casino Drive, an early 7-2 second choice, was having the hoof on a hind leg treated with ice and heat.
Re: Belmont Stakes Raceday
Belmont is not a good race for bettors
Expected dominance of Big Brown makes it difficult to put together a profitable combination.
Because of what is on the line, the 140th Belmont Stakes is the most anticipated race of 2008.
With a victory, Big Brown, who has been dominant in the Kentucky Derby and Preakness, will become the sport's first Triple Crown winner since Affirmed in 1978.
But the Belmont is not a good betting race. Unless, of course, one believes Big Brown is vulnerable.
The recent issue with one of Big Brown's hoofs caused concern for some, but the undefeated colt's solid work on Tuesday seemed to dispel any fears.
The feeling is Big Brown will do his thing Saturday in Elmont, N.Y., succeeding where Silver Charm, Real Quiet, Charismatic, War Emblem, Funny Cide and Smarty Jones have failed since 1997.
The rail is not the spot Rick Dutrow, Jr. wanted, but at the post position draw on Wednesday morning Big Brown's trainer said that would not cause his horse to lose.
If Big Brown runs anywhere close to the way he did in Kentucky or Maryland, the rest are again running for second.
His instant acceleration in the Preakness was breathtaking, and the race could not have taken much out of him given how jockey Kent Desormeaux had him under wraps nearing the wire.
Big Brown's logical partner in an exacta pick is the undefeated Casino Drive, but the choice here is Denis Of Cork.
A son of Harlan's Holiday who skipped the Preakness after finishing third in the Kentucky Derby, Denis Of Cork acts like a colt who wants 1 1/2 miles. He should be finishing while others in the Belmont may be looking for a place to lie down in the final quarter of a mile.
He's also reunited with jockey Robby Albarado, who was aboard for Denis Of Cork's most impressive performance -- a 2 1/4 -length victory in the Southwest at Oaklawn Park in Hot Springs, Ark. on Feb. 18.
Casino Drive, who merits respect, is out of Better Than Honour -- a dam who has produced consecutive Belmont winners, Jazil and Rags To Riches -- so 12 furlongs will not bother the son of former horse of the year Mineshaft.
However, what Casino Drive's connections described as a minor problem with his left hind leg surfaced on Friday morning, so his status for the Belmont won't be decided until Saturday morning.
With the uncertainty, it might be best for exotics players to toss Casino Drive, who, if he runs, will be seeking to become only the third horse to win the Belmont in his only his third career start. The last to do it was Prince Eugene 95 years ago.
Casino Drive certainly has the right jockey to play Belmont spoiler. Edgar Prado guided Birdstone to a 36-1 upset of Smarty Jones four years ago and was also the rider for 70-1 Sarava in 2002 when War Emblem was seeking his place in history.
As for the others, Ready's Echo may be best as a late-running sprinter, Macho Again and Icabad Crane couldn't get close to the favorite in the Preakness, and Anak Nakal, Da'Tara and the maiden Guadalcanal are overmatched.
Tale Of Ekati is somewhat interesting given his two-for-two record at Belmont, but he will have to improve significantly off his lone win of 2008, a slow score in the Wood Memorial on April 5 at Aqueduct.
Still, the son of Tale Of The Cat has trained well in recent weeks for owner-breeder Charles Fipke and trainer Barclay Tagg. Maybe Tagg can do to Dutrow what Bobby Frankel, a good friend of Dutrow's, did to him in 2003.
Tagg was the trainer of Funny Cide, whose bid for a Triple Crown was dashed when the Frankel-trained Empire Maker won the Belmont over a sloppy surface.
Re: Belmont Stakes Raceday
A Different Kind of Bottom Line
By Anthony Stabile
For those of you that thought you'd be reading the same Bottom Line format I used for both the Kentucky Derby and Preakness, I apologize for changing it up on you. I promise to make it up to you if you continue reading.
Since he crossed the wire under wraps in the Preakness, I'm sure you wish you had a dollar for every time you've heard or read that Big Brown can't lose the Belmont on Saturday. You've listened to his trainer, Rick Dutrow Jr., use words like "forgone conclusion," when it comes to Big Brown wrapping up the Triple Crown and joining the eleven others in the pantheon of Triple Crown greats.
I don't know about you, but I'm sick of it. And you won't be reading any of that in this column because I'm here to tell you that Big Brown will not win this Saturday's Belmont Stakes. In fact, I think there is a good chance he'll be off the board. Here's why.
The Quarter Crack – I know Dutrow and foot specialist Ian McKinley are saying there is nothing to worry about, but what are they supposed to say? Imagine how foolish it would appear if they were to say "this could really be a problem, but we're running him anyway," especially on the heels of the Eight Belles catastrophe in the Derby? I'm not saying the horse is in grave danger because they wouldn't be running him if he would. At least that's what I'd like to think. But, to say it won't affect his performance is nonsense. And, let's not forget that this is a new quarter crack on his other foot. The rain that fell in Louisville before the Derby and in Baltimore before the Preakness was the best thing that ever happened to Big Brown because of instead of worrying about scraping the courses down to illicit faster times on the two big days, the maintenance crews were far more concerned with getting the courses in manageable condition. That probably won't be the case at Belmont as NYRA is notorious for jazzing these tracks up on big days.
The Pace Scenario – After getting perfect set ups in the first two jewels, Big Brown will now break from the rail and you have to assume he'll use his speed in order to maintain his position early on. I think Desormeaux is putting him on the lead and with a horse like Da' Tara who is seemingly in the race to insure a quick pace it could be Triple Crown suicide. I just don't see him rating from the rail and working out a good trip if he does.
Casino Drive – Should he manage to work out a stalking trip, he won't be the only one as the Japanese wonder horse should be placed right along side of Big Brown. It could be even worse for Big Brown should Casino Drive take the initiative and grab the early lead. Big Brown has never had to pass a horse with the ability and potential of Casino Drive. No one really knows how he'll react.
The Distance – The 1 ½ miles of "the Test of the Champion" will likely be the most grueling race in most of these horses respective careers. Not only is Big Brown not bred particularly well to handle the distance, others in here are. Casino Drive is a half brother to the last two Belmont winners, Jazil and Rags to Riches, Tale of Ekati's pedigree is laden with stamina, especially on the dam side and longshot Anak Nakal is sired by 1998 Belmont winner Victory Gallop who upset Real Quiet in his Triple Crown bid.
Kent J. Desormeaux - Let's call a spade a spade – the guy cost Real Quiet the Triple Crown. It's as simple as that. He's been known to get overly aggressive and make knucklehead moves from time to time and who knows what he's going to do with Big Brown once the gate opens? The answer: not even him.
Karma – Can you really talk this much smack and win? Doesn't Dutrow know that the Racing Gods hate braggarts? I mean, I wouldn't mind if this was all by design but if the allowance race he won in his first start this year didn't get rained off the turf, Big Brown would be 1-5 in the Hill Prince at Belmont on Friday. Quarter cracks aside, everything has pretty much gone perfectly for these guys. In this business, that ends at some point. I think Saturday is the perfect day for it.
There is a reason 10 horses have failed in their attempts to win the Triple Crown the past 30 years – IT'S HARD!!! In my opinion, you need to have eight perfect weeks, starting three weeks before the Derby and ending when you cross the wire in the Belmont. Big Brown hasn't and his misfortune has befallen him at the most inopportune of times. Come Saturday, Big Brown will turn into Big Frown for there'll be no Triple Crown.
And that's the bottom line.
Re: Belmont Stakes Raceday
Betting on Belmont: Glantz picks his favorites
It's been 30 years since Affirmed won the Belmont Stakes and the Triple Crown.
Most recently, it was Smarty Jones that won the Kentucky Derby and Preakness Stakes but failed to capture the third leg.
I can remember the excitement in the Las Vegas books as Smarty Jones appeared to have the race in hand when Birdstone came into the picture in the stretch and the silence of the crowd became apparent. The only words that could be heard were "hurry, Smarty" and "c'mon, Smarty." Of course we know the outcome; Birdsong caught Smarty at the 16th pole and went on to win the Belmont by a single length.
The problem with the Belmont is that while a horse seems to be a world-beater after victories in the Kentucky Derby and the Preakness Stakes, the final leg of the Triple Crown is always the most difficult normally because the first two races took so much out of the winning horse and/or because he's coming back in just three weeks facing fresh horses in the longest test of the three.
So how will Big Brown react to a possible off-track (30 percent chance of thunderstorms)? Will that quarter crack become a factor? While the one-hole has produced the most winners in past Belmont Stakes (23), will B.B. be affected by the maiden next to him if Guadalcanal acts up either in the gate or perhaps with a poor break?
Well, let's take a look at this field in the 140th running of the Belmont Stakes by post position and their morning line odds:
1) Big Brown (2-5) – B.B. has won all his five races by a combined 39 lengths but most importantly, he won the Kentucky Derby pulling away by 4 ¾ lengths and the Preakness Stakes by 5 ¼ lengths. In the Preakness, his awesome move in the straightaway was a sight to see. He had a bullet drill prior to the Derby and in his final workout prior to the Belmont he turned in another. Big Brown has yet to be tested so we really don't know how good he is, but he's without question the horse to beat.
2) Guadalcanal (50-1) – Appears to like the distance but this one hasn't been able to beat maidens in five tries. While Guadalcanal doesn't belong here, let's just hope he breaks clean and doesn't bother Big Brown.
3) Macho Again (20-1) – Ran second to Big Brown in the Preakness after capturing the 7 ½ F Derby Trial at Churchill the last week in April. Macho's recent works haven't impressed but he has a chance for a piece.
4) Denis of Cork (12-1) – Denis was the even-money favorite in the Illinois Derby but failed to fire than ran a surprising third in the Kentucky Derby. His last work was considerably less than impressive but he's fresh and after breaking 20th in the Derby, his closing kick can't be ignored.
5) Casino Drive (7-2) – The real unknown here. Casino Drive has had only two races, one in Japan breaking his maiden by 11 ½ lengths and then his impressive 5 ¾ length win in the Peter Pan Stakes over this course in May. Casino's Dam, Better Than Honour, was also the mom to Jazil and Rags to Riches, the last two Belmont Stakes winners. No matter what Dudrow says, Casino Drive can't be ignored.
6) Da'Tara (30-1) – This one finished ninth, 23 ½ lengths behind Big Brown, in the Florida Derby earlier in the year and fifth, five lengths in back of Macho Again in the Derby Trial. His only win came in a maiden race. An in-the-money finish would be a surprise.
7) Tale of Ekati (20-1) – Tale of Ekati has a win here in the Grade 2 Futurity last year. Adding to his resume is his win in the Wood Memorial when he held off War Pass in April. He finished a distant fourth in the Kentucky Derby but, like Denis of Cork, is well rested. Unlike D of C, Ekati has put in three pretty good works. Must consider.
8) Anak Nakal (30-1) – After two wins and a second in his three races last year, Anak Nakal has failed to show up in his four races this year. Perhaps his 45-1 odds in the Wood and 54-1 odds in the Derby tells the tale.
9) Ready's Echo (30-1) – Another whose only win came in a maiden race but Echo did finish a respectable third (by 6 ¼ lengths) in the Peter Pan against Casino Drive.
10) Icabad Crane (20-1) – Watch the tote board on this one as Icabad has been receiving some positive press. He finished third behind Big Brown and Macho Again but didn't have a clear trip in the Preakness. Must be respected.
Glantz's Belmont Selections:
1. Big Brown
2. Casino Drive
3. Icabad Crane
4. Tale of Ekati
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You won't be able to dismiss this by usual means (escape or click button), but you can close it programatically based on user choices or actions.
"My save the date cards look beautiful! I loved that I could personalize it by changing the colours of the card to my exact wedding colours. Also, the customer service was excellent and shipping was also super fast!"
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Click here for the iOS version, and here for Android.
What's so good?
By Jason Grishkoff | Dec 12, 2012 | Total plays: 12,022
In this case, that's likely owing to the fact that John Hill is doing the mixing -- his prior work includes songs for Rihanna, Santigold and M.I.A. Certainly this sound is a bit of a departure from those female powerhouses, but it seems to work quite well.
Wavves of course is one-man-band Nathan Williams. "Sail To The Sun" will be on released on his next album, to be available in the first part of 2013.


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December 12, 2013
(15 Sept. 2012) --- Expedition 31/32 and Expedition 32/33 crew members pose for a group portrait in the International Space Station's Zvezda Service Module following the ceremony of Changing-of-Command from Expedition 32 to Expedition 33. Pictured on the front row are NASA astronaut Sunita Williams, Expedition 33 commander; and Russian cosmonaut Gennady Padalka, Expedition 32 commander. Pictured on the back row (from the left) are Japan Aerospace Exploration Agency astronaut Aki Hoshide and Russian cosmonaut Yuri Malenchenko, both Expedition 33 flight engineers; NASA astronaut Joe Acaba and Russian cosmonaut Sergei Revin, both Expedition 32 flight engineers.
Topics: Expedition 32, Human spaceflight, Spaceflight, Expedition 33, Expedition 31, Sergei Revin, Akihiko Hoshide, Expedition 9, Gennady Padalka, STS-106, Yuri Malenchenko


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"Hacking is about exploring the limits of what's possible."
Echoing Green Blog
Take a role in the social impact movement by applying your talent to a job that is right for you, and good for the world.
Eighteen Echoing Green Fellows were recognized on Forbes' 2015 "30 under 30" list for their innovative work on tackling the world's biggest problems.
What happens when 150 Echoing Green Fellows live, breathe, and debate social change amidst the hustle and bustle of one of the world's largest cities – and what lessons can you incorporate into your life?
Fellows respond to the President's actions with some hope, and acknowledgement of the distance yet to overcome.
Take a role in the social impact movement by applying your talent to a job that is right for you, and good for the world.
Echoing Green's approach to supporting groundbreaking, early-stage social entrepreneurs runs much deeper than a two-year stipend and health insurance.
Last Mile Health is developing a clear and comprehensive strategy to combat Ebola, and to strengthen the Liberian health system for the long term.
Part pragmatism, part political theater, Bren is drawing attention to the plight, and the opportunities, of the sea.
Priya Haji influenced a generation of social entrepreneurs through her passion, warmth, strength, and idealism. We will miss her.


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Basketball Player Or Wizard?
Michigan's Mitch McGary might be the breakout player of the 2013 NCAA Tournament. And based on this play, he also might be magic.
1. Mitch McGary has been dominant during Michigan's run to the Final Four — through four games, he's averaging 18 points and 12 rebounds. But during today's Elite Eight win against Florida, he pulled off his most remarkable play yet.
2. Let's take a closer look. McGary shows tremendous concentration, following the ball with his eyes while flying out of bounds. It's an amazing play, particularly for someone who's six-foot-ten.
3. Of course, this superhuman coordination begs one specific question: is Mitch McGary a wizard?
4. …
5. …
7. I guess that's settled.
Preview Your Response


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HSBC Trinkaus & Burkhardt AG (HSBC Trinkaus) is a financial service provider, based in Germany. The company operates through Corporate Client, Private Banking, and Institutional Client sectors. Its Corporate Client sector offers professional services, such as lending and deposit products, and domestic and foreign payment transactions service to large and medium-sized companies. The company's Private Banking sector provides advisory and management services for private portfolios. Its Institutional Client sector offers traditional and modern investments, solutions, and refinancing instruments to institutional clients, including insurance companies, pension and investment funds, and banks. The company has its presence in Baden-Baden, Berlin, Dusseldorf, Frankfurt/Main, Hamburg, Cologne, Munich, Stuttgart, and Luxembourg. It operates in more than 80 countries, five continents. HSBC Trinkaus is headquartered in
Author:Bill Thompsone-mailWeb: www.fastmr.comPhone: 18008448156


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Professor John Reilly, of Strathclyde University, has joined an international panel help seek solutions to the causes and problems associated with the condition.
Professor Reilly, of the University's School of Psychological Sciences & Health, is one of only 17 members of the working group reporting to WHO's Director General on science and evidence for ending childhood obesity.
The group will be assessing research and will report back to the director general, Dr Margaret Chan, to the World Health Assembly in 2015.
WHO statistics indicate that at least 40 million children worldwide under the age of five were classed as obese in 2012.
The total is projected to rise to at least 70 million by 2025.


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Latest Geothermal Resources Council Stories
Special Deals for Early Birds and Students at Geothermal Energy Event of the Year (Portland, Oregon, USA) (PRWEB) July 16, 2014 Registration is now open
Major names in the international geothermal community and regional and national politics will address the geothermal energy industry at the GRC Annual Meeting in Portland, Oregon, USA.
$15,000 in Scholarship Awards will be presented at the 2014 GRC Annual Meeting in Portland, Oregon, USA. Davis, California, USA (PRWEB) June 23, 2014
Popular networking event for the geothermal energy industry on Sept.
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Popular Events Before and After the GRC Annual Meeting & GEA Expo in Portland, Oregon Portland, Oregon, USA (PRWEB) May 23, 2014 The Geothermal Resources
The Geothermal Resources Council will present a two-day course in Jakarta before the 2nd Indonesia International Geothermal Conference and Exhibition. Davis,
The Geothermal Resources Council backs the plan to restore the inland sea using revenue from new geothermal energy development. Davis, California, USA (PRWEB)
The 33rd Annual Award winners will be announced at the Geothermal Resources Council Annual Meeting in Portland, Oregon, USA. Davis, California (PRWEB) March
Hotel room reservations can now be made for the geothermal event of the year in Portland, Oregon, September 29 – October 2. Davis, California, USA (PRWEB)
- An uxorious, effeminate, or spiritless man.
- A timorous, cowardly fellow.


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Comment: Re:Either of the poles woulc cause this effect (Score 1) 495
If you start a mile north of the South Pole, walk a mile south, then you cannot walk west, so it still fails.
Also, the North Pole isn't ice-free all year long. (I've not been keeping up with how much (if it has happened yet) it is ice-free during a year, but it's certainly not the whole year. Yet.)


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Now is the time to make sure your website is ready to turn interactions into transactions!
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Join Magento, along with our partners One Pica, SLI, Kount, Peer1, and Kiosked to gain valuable tips on how to ensure that your site can deliver the best possible results during peak season activity and beyond.
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Sorry! The position you're looking for is no longer available. Check out other Fashion internships in New York, NY below.
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Specifying Startup Settings
Select the Startup tab.
Use the Startup tab to customize the default values that appear on the signon screen.
Signon Defaults
- Database Type
Select the database type to appear as a default on the PeopleSoft Signon dialog box. Select Application Server to sign in to an application server instead of a database. To enable users to change their database type selection in the signon dialog box, you must select theDatabase Type option in theUser Can Override group.
Note: When you select Application Server from theDatabase Type drop-down list, theServer Name andDatabase Namefields are disabled. The system obtains these values from the application server.
- Application Server Name
If you selected Application Server from theDatabase Type drop-down list, specify the application server's name in this field. You must have already configured your application server and registered it on the Profile tab.
- Server Name
Enter the name of the default database server. This parameter is only enabled for Informix, Sybase, and Microsoft SQL Server, and refers to the instance to which the user connects.
For Informix, enter the server name in lowercase.
- Database Name
Enter a default database name. You can choose any valid PeopleSoft database name. As with the database type, you must select the appropriate option in the User Can Override group to enable users to override the default database name selection when they sign in.
- User ID
Specify the default user ID to sign in to PeopleSoft.
You can use the user ID in conjunction with a PSUSER module containing a user-defined sign-in process. The PSUSER code, if present, can evaluate and modify the user ID value before you attempt to sign in to the selected database.
- Connect ID andConnect Password
PeopleSoft uses the connect ID for the initial connection to the database. Use the Connect Password field to define a default connect ID password.
Note: The connect ID edit box must contain a value, or the user can't sign in to the system in a two-tier environment.
See PeopleSoft Authorization IDs.
See PeopleTools Installation Guide for Your Database Platform.
Numeric Keypad - Enter Key Tabs to Next Field
In Microsoft Windows applications, pressing the Enter key in a dialog box selects the default action button. For example, in the PeopleSoft Signon dialog box, pressing Enter is the same as clicking theOK button. Selecting theNumeric keypad check box overrides this default behavior for the Enter key on the numeric keypad; instead of selecting the action button, pressing the Enter key moves the cursor to the next field in the dialog box.
Note: This check box affects the Enter key on the numeric keypad, but not the Enter key on the main keyboard.
User Can Override
Some PeopleSoft sites use multiple database types and names. Using the check boxes in the User Can Override group box, you can enable users to enter a database type, database name, or user ID other than the default provided at sign-in. In most cases, you use these controls to prevent users from attempting to sign in onto any database other than the default.
- Database Type
When selected, users can choose a database other than the default. Selecting this check box selects the Database Name andUser ID options automatically. You cannot clearDatabase Name orUser ID without first clearingDatabase Type. When configuring a workstation to connect in both two-tier and three-tier, you must select this box. The user needs to specify a two-tier or three-tier connection from the PeopleSoft Signon dialog box.
- Database Name
When selected, the User ID check box is automatically selected, although you can clear it. To clearDatabase Name, you must clear theDatabase Type check box.
- User ID
Select to enable users to users override only the user ID submitted at when they sign in. You cannot clear User ID ifDatabase Type is selected.
Cache Files Directory
Enter a parent directory to hold your client cache file directories. The directory you specify must be writable for a user with Standard User privileges. For example, %USERPROFILE%\psft\pt\client\cache.
Cache files store database object information locally and are automatically downloaded the first time you open a PeopleSoft database object. They are also downloaded automatically if the master copy of the object on the database server has changed. For each PeopleSoft database to which you connect, the system creates a single child cache files directory to store the cache files for that database.
Clicking Purge Cache Directories brings up a dialog box with your existing cache file directories.
You can select a single directory and click Delete, or you can clickDelete All to remove all directories. If a cache file directory is missing (after you delete it), the system automatically rebuilds it the next time that cache files are downloaded. After you delete the appropriate cache directory, clickClose to return to the Startup tab.


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Govt to unveil `major initiative' in next 10 days
Our Bureau
New DelhiMay 22The Prime Minister, Dr Manmohan Singh, on Tuesday said that the country was headed for a fifth successive year of 8.5 per cent plus growth, made possible by investment rates hitting an all-time high of 35 per cent of the national income.
In a report marking the completion of three years by the United Progressive Alliance (UPA) Government at the Centre, the Prime Minister sought to allay concerns that the present growth phase had left out vast sections of the population or accentuated regional imbalances.
Stating that the UPA Government was promoting an "inclusive growth" strategy, Dr Singh said that this had been done through stepping up investments in rural infrastructure and agriculture, increasing credit availability to farmers and offering them remunerative price for their produce, besides putting in place the National Rural Employment Guarantee Programme and enhancing public spends on education and healthcare.
He said the Government would be unveiling a "major initiative" in the next 10 days, which will not only enhance the total public investment in agriculture, but will also enable States to resolve problems of agriculture through comprehensive localised plans.
"Through Bharat Nirman, we expect to invest Rs 1,74,000 crore in rural roads, housing, drinking water supply, irrigation, electrification and telecommunication," he said.
Further, the budgetary allocation for education has been nearly trebled in three years to Rs 32,000 crore in 2007-08, while that for health and family welfare more than doubled to Rs 17,560 crore.
In agriculture and rural development, funding has been stepped up to over Rs 60,000 crore this fiscal, he added.
The Prime Minister said that the massive increase in public spending had not led to the Government reneging on its commitment to fiscal discipline.
While admitting that the growth processes "unleashed" by his Government had put "some pressure" on prices, he maintained that "credible steps" had been taken to control inflation.


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Does anyone have an Elna? Do you like it? I have looked all over the web for reviews and can not find any. I a looking at a floor model of the elna 654, its oeen onthe floor since Christmas. Has the full warrenty and 2 years of services and classes included in the $429 price. Normal retail is $599. Any recommendations and thoughts woudl be apprecited@! I am new to serging -
I have two Elna sewing machines and love them both, however they are sewing machines, not sergers. One is new to me, the other I've had for years with no troubles. My mom has had an Elna for as long as I remember.
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Today, thousands of people return for the second day of the 2011 ACORD LOMA Insurance Systems Forum, bringing together the best in insurance and technology. This year, Microsoft attends the conference (booth #403) with six key partners that offer innovative solutions to the industry: Wipro, Accenture, Hyland, Neudesic, VUE Software and Customer Effective.
As we see it, technology is evolving at a blurring rate and is changing the way insurance firms operate, communicate and relate to their customers and each other. Just as Cindy Saccocia wrote in a recent post, we take note of some key trends that will be important for this industry:
Together with our partners, Microsoft will be showing ACORD attendees just how these ideas can be put in place within their own organizations. Last night, Colin Cole, Industry Technology Strategist for Insurance, US Financial Services Group, discussed the architecture behind Microsoft's public cloud platform, Azure, and trained attendees on how to begin to incorporate leverage a public cloud platform into their software offerings. As he shared, cloud technology gives insurance companies great flexibility in moving data from the back office to the PC, to mobile devices and across continents in an increasingly borderless world.
Microsoft and our partners are also participating in the following events:
We look forward to sharing more insights gathered at this year's conference. For additional information on industry trends and Microsoft's participation at ACORD, please visit our Insurance Industry website.