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|Nov 18, 1997
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|Christopher L. Baszczynski, Lesek Alexander Lyznik, William J. Gordon-Kamm, Xueni Guan
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|Pioneer Hi-Bred International, Inc.
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This application is a Continuation of U.S. patent application Ser. No. 11/400,293 (now U.S. Pat. No. 7,364,902), filed Apr. 7, 2006, which is a Continuation of U.S. patent application Ser. No. 10/444,664 (now U.S. Pat. No. 7,179,599), filed May 23, 2003, which is a Continuation of U.S. patent application Ser. No. 09/411,826 (now U.S. Pat. No. 6,664,108), filed Oct. 1, 1999, which is a Divisional of U.S. Pat. No. 6,300,545 (U.S. patent application Ser. No. 09/193,475, filed Nov. 17, 1998), which claims the benefit of U.S. Provisional Application Ser. No. 60/099,461, filed Sep. 8, 1998, U.S. Provisional Application Ser. No. 60/065,627, filed Nov. 18, 1997, U.S. Provisional Application Ser. No. 60/065,613, filed Nov. 18, 1997, all of which are herein incorporated by reference.
The present invention relates to plant molecular biology. More specifically, the invention relates to methods and compositions for increasing the efficiency of excision of viral replicon from T-DNA that is transferred to a plant by agroinfection.
Agrobacterium harboring a Ti or a Ri plasmid can efficiently transfer a portion of these plasmids, the T-DNA, into plant cells at the site of a wound. Transfer of the T-DNA into the plant cell is induced by signal compounds present at the site of a plant wound and requires T-DNA border sequences at both ends of the T-DNA and trans-acting virulence gene products (vir) encoded by the Ti or Ri plasmid. The transferred T-DNA is then targeted to the nucleus and integrated into the plant genome. A tumor, crown gall, forms at the site of inoculation in most dicotyledonous plants.
Tumor formation results from the expression of T-DNA oncogenes encoding the growth factors auxin and cytokinin which promote plant cell proliferation. In order for these oncogenes to be expressed, the T-DNA must first integrate into the genome of the plant. Tumor formation is limited to dicotyledonous plants because the T-DNA, while transferred into the cells of monocots, is either not normally integrated into the genome of monocotyledonous plants or is integrated and silenced.
By inserting a viral genome into the T-DNA, Agrobacterium can be used to mediate the viral infection of plants. Following transfer of the T-DNA to the plant cell, excision of the viral genome from the T-DNA (mobilization) is required for successful viral infection. This Agrobacterium-mediated method for introducing a virus into a plant host is known as agroinfection (for a review, see Grimsley, "Agroinfection" pp. 325-342, in Methods in Molecular Biology, vol 44: Agrobacterium Protocols, ed. Gartland and Davey, Humana Press, Inc., Totowa, N.J.; and Grimsley (1990) Physiol. Plant. 79:147-153, the contents of which are incorporated herein by reference). Upon entry into the plant cell nucleus, a unit length circular viral genome that is capable of initiating systemic infection is mobilized from the T-DNA. Integration of the T-DNA into the plant genome is not required for this event. Two non-exclusive mechanisms, intramolecular homologous recombination and replicative release, have been proposed for this release of circular viral genomes or replication intermediates from T-DNA. The replicative release of viral genomes by the rolling-circle replication mechanism has been demonstrated for the mobilization of geminivirus genomes from T-DNA (Stenger et al. (1991) Proc. Natl. Acad. Sci. 88:8029-8033). Release of geminivirus via homologous recombination between tandemly repeated genomes has also been demonstrated (Lazarowitz et al. (1989) EMBO J. 8:1023-1032).
Release by either of the above mechanisms requires the presence of tandem directly repeated copies of the viral replicon in the T-DNA. A circular viral replicon may be excised from the T-DNA by intramolecular homologous recombination between the repeated genomes. For replicative release, two origin of replication sequences must be present to initiate and complete the replication process. Both mechanisms of mobilization are complex biochemical processes that may be attenuated by a number of factors which in turn affect the efficiency of viral excision. Furthermore, tandem dimers of viral DNA are often difficult to construct and are unstable in recombination proficient host cells.
Agroinfection has been reported in a number of publications as a successful method for inducing systemic viral infections in plant cells, including monocotyledonous plants such as maize (Heath et al. (1997) Mol. Plant-Microbe Interact. 10:221-227, Grimsley et al. (1989) Mol. Gen. Genet. 217:309-316, the contents of which are incorporated herein by reference). In many instances, particularly when naked viral nucleic acid is non-infectious, agroinfection is the only way of transforming a plant with cloned viral DNA. Even where naked viral nucleic acid is infectious, agroinfection is frequently used because it is relatively efficient and does not require the production of large amounts of plasmid or viral DNA. Agroinfection has been used to study viral replication and recombination, in the investigation of viral gene functions, for the production of autonomously replicating viral vectors, for transient expression of genes inserted into T-DNA, for integration of DNA into a plant genome, for production of virus resistant plants, for the study of transposable elements and for the determination of tissue-specific susceptibility to T-DNA transfer.
The development of plant virus gene vectors for expression of foreign genes in plants provides a means to provide high levels of gene expression within a short time. The benefits of virus-based transient RNA and DNA replicons include rapid and convenient engineering coupled with flexibility for expeditious application in various plant species. In this manner, autonomously replicating viruses offer numerous advantages for use as vehicles for transient expression of foreign genes, including their characteristic high levels of multiplication and concomitant levels of transient gene expression. Accordingly, it would be beneficial to provide methods that facilitate the construction of vectors for agroinfection, provide flexibility in designing viral vectors for genetic transformation of plant cells and increase the efficiency of mobilization of viral replicons from T-DNA and the copy number of a DNA sequence of interest associated with the viral replicon.
The present invention accomplishes these objectives by providing methods and compositions for the mobilization of viral replicon from T-DNA via site-specific recombination systems. Site-specific recombination-mediated excision of DNA fragments from chromosomal or extrachromosomal DNA molecules has been described for a number of site-specific recombination systems and plant species. See Russell et al. (1992) Mol. Gen. Genet. 234:49-59; Lyznik et al. (1996) Nucleic Acids Res. 24:3784-3789; and Dale et al. (1991) Proc. Natl. Acad. Sci. 88:10558-10562 the contents of which are incorporated by reference. However, mobilization of viral vectors from T-DNA via site-specific recombination has not previously been applied to agro-mediated transformation.
The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise Agrobacterium-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell.
The compositions of the invention comprise nucleic acids, such as T-DNAs containing a viral DNA flanked by directly repeated target sites for a site-specific recombinase. The nucleic acids of the invention may additionally contain expression cassettes encoding the cognate site-specific recombinase for the target sites flanking the viral genome. The compositions of the invention further comprise Agrobacterium containing the nucleic acids of the invention.
The compositions and methods of the invention have use in providing high copy numbers of a DNA of interest for transient expression or for integration into a plant chromosome, in simplifying the construction and stable maintenance of vectors for agro-mediated transformation of plants and in increasing the efficiency of agroinfection.
The invention is directed to methods and compositions for the mobilization of viral DNA from T-DNA. In the methods of the invention, a viral replicon flanked by directly repeated target sites for a site-specific recombinase is inserted into a T-DNA carried by an Agrobacterium replicon. The T-DNA is transferred into the cell of a plant by agro-mediated transfer. Expression of the site-specific recombinase in the infected plant cell will result in excision of a circular viral replicon from the transferred T-DNA (
Thus, the invention is directed to a method for mobilizing a viral replicon from a T-DNA, comprising:
a) providing an Agrobacterium replicon having a T-DNA, wherein said T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase; and
b) infecting a cell of a plant with an Agrobacterium carrying said Agrobacterium replicon under conditions that allow transfer of said T-DNA and expression of said recombinase in said cell;
wherein said cell, said T-DNA, or said viral replicon contains a nucleotide sequence encoding said recombinase or an active fragment or variant thereof, and said nucleotide sequence is operably linked to a promoter that drives expression in said cell.
If a DNA of interest has been inserted into the viral replicon, or between the viral replicon and a target site for a site-specific recombinase, the DNA of interest will also be replicated to high copy. The high copy number of the DNA of interest increases the efficiency of integration of this DNA into the genome, or the level of transient expression of a gene encoded by the DNA of interest. Thus, in another aspect, the invention provides a method for providing to a plant cell a plurality of copies of a DNA sequence of interest, comprising:
a) providing an Agrobacterium replicon having a T-DNA, wherein said T-DNA contains in a 5′ to 3′ or 3′ to 5′ orientation, a first target site for a site-specific recombinase, a viral replicon, said DNA sequence of interest, and a second target site for said recombinase in direct repeat with said first target site, wherein said first and second target sites are identical; and
b) infecting a cell of a plant with an Agrobacterium carrying said Agrobacterium replicon under conditions that allow transfer of said T-DNA and expression of said recombinase in said cell; wherein said cell, said T-DNA, or said viral replicon contains a nucleotide sequence encoding said recombinase or an active fragment or variant thereof, and said nucleotide sequence is operably linked to a promoter that drives expression in said cell.
The methods of the invention are useful for providing a high copy number of a DNA of interest for site-specific integration into a plant chromosome. In this case, the plant chromosome will contain one or more target sites for a site-specific recombinase. Depending on the substrates, the recombination event could consist of a single or a double cross-over event. In the simplest case, the plant chromosome and the excised viral replicon each contain a single target site. Site-specific recombination between these two target sites results in the insertion of the viral replicon and any associated DNA of interest into the plant chromosome.
The inclusion of a non-identical target site between the viral replicon and the DNA of interest allows integration of the DNA of interest into a plant genome having a corresponding target site, without concomitant insertion of the viral replicon. In this method, the distal ends of the viral replicon and the DNA of interest are flanked by identical target sites. Recombination between the identical target sites results in excision of a circular viral replicon containing the DNA of interest flanked by non-identical target sites for the recombinase (
Thus, in a further aspect, the invention provides a method for providing to a plant cell a plurality of copies of a DNA sequence of interest flanked by nonidentical target sites for a site-specific recombinase, comprising:
a) providing an Agrobacterium replicon having a T-DNA, wherein said T-DNA contains in a 5′ to 3′ or 3′ to 5′ orientation, a first target site for said recombinase, a viral replicon, a second target site for said recombinase, said DNA sequence of interest, and a third target site for said recombinase, wherein said first and third target sites are directly repeated and identical with respect to each other, and said second target site is nonidentical to said first and third target sites; and
b) infecting a cell of a plant with an Agrobacterium carrying said Agrobacterium replicon under conditions that allow transfer of said T-DNA and expression of said recombinase in said cell;
wherein said cell, said T-DNA, or said viral replicon contains a nucleotide sequence encoding said or an active fragment or variant thereof, and said nucleotide sequence is operably linked to a promoter that drives expression in said cell.
The compositions of the invention comprise T-DNAs containing a viral replicon flanked by directly repeated target sites for a site-specific recombinase. In another aspect, the compositions of the invention comprise a T-DNA containing in a 5′ to 3′ or 3′ to 5′ orientation, a first target site for said recombinase, a viral replicon, a second target site for said recombinase, said DNA sequence of interest, and a third target site for said recombinase, wherein said first and third target sites are directly repeated and identical with respect to each other, and said second target site is nonidentical to said first and third target sites. The T-DNAs of the invention may further comprise nucleotide sequence encoding a site-specific recombinase, wherein the nucleotide sequence is operably linked to a promoter that drives expression in a plant cell. The compositions of the invention further comprise replicons containing these T-DNAs and Agrobacterium containing these replicons.
By "mobilization of a viral replicon" is meant excision of a viral replicon from a T-DNA sequence after it has been transferred to a plant cell. In the methods of the invention, mobilization of viral DNA is accomplished by conservative site-specific recombination between directly repeated target sites flanking the viral DNA. In one embodiment, the resulting product is a circularized viral DNA containing one copy of the target site (see
By "viral replicon" is meant double-stranded DNA from a virus having a double stranded DNA genome or replication intermediate. The excised viral DNA is capable of acting as a replicon or replication intermediate, either independently, or with factors supplied in trans. The viral DNA may or may not encode infectious viral particles and furthermore may contain insertions, deletions, substitutions, rearrangements or other modifications. The viral DNA may contain heterologous DNA. In this case, heterologous DNA refers to any non-viral DNA or DNA from a different virus. For example, the heterologous DNA may comprise an expression cassette for a protein or RNA of interest.
Viral replicons suitable for use in the methods and compositions of the invention include those of viruses having a circular DNA genome or replication intermediate, such as: Abuitilon mosaic virus (AbMV), African cassaya mosaic virus (ACMV), banana streak virus (BSV), bean dwarf mosaic (BDMV), bean golden mosaic virus (BGMV), beet curly top virus (BCTV), beet western yellows virus (BWYV) and other luteoviruses, cassaya latent virus (CLV), carnation etched virus (CERV), cauliflower mosaic virus (CaMV), chloris striate mosaic (CSMV), commelina yellow mottle virus (CoYMV), cucumber mosaic virus (CMV), dahlia mosaic virus (DaMV), digitaria streak virus (DSV), figwort mosaic virus (FMV), hop stunt viroid (HSV), maize streak virus (MSV), mirabilias mosaic virus (MMV), miscanthus streak virus (MiSV), potato stunt tuber virus (PSTV), panicum streak virus (PSV), potato yellow mosaic virus (PYMV), rice tungro bacilliform virus (RTBV), soybean chlorotic mottle virus (SoyCMV), squash leaf curl virus (SqLCV), strawberry vein banding virus (SVBV), sugarcane streak virus (SSV), thistle mottle virus (ThMV), tobacco mosaic virus (TMV), tomato golden mosaic virus (TMGV), tomato mottle virus (TMoV), tobacco ringspot virus (TobRV), tobacco yellow dwarf virus (TobYDV), tomato leaf curl virus (TLCV), tomato yellow leaf curl virus (TYLCV), tomato yellow leaf curl virus-Thialand (TYLCV-t) and wheat dwarf virus (WDV) and derivatives thereof. Preferably the viral replicon is from MSV, WDV, TGMV or TMV.
By "T-DNA" is meant the T-DNA of an Agrobacterium tumefaciens Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid, or a derivative thereof. The T-DNA may comprise an entire T-DNA, but need only comprise the minimal sequences required in cis for transfer (i.e., the right and the left T-DNA border sequences). The T-DNAs of the invention have inserted into them, anywhere between the right and left border sequences, a viral DNA flanked by target sites for a site-specific recombinase. The T-DNA may contain deletions, substitutions and/or additional insertions of DNA other than the viral genome and target sites. Preferably the T-DNA contains an expression cassette for the cognate recombinase of the target sites flanking the viral DNA. The sequences encoding factors required in trans for transfer of the T-DNA into a plant cell, such as vir genes, may be inserted into the T-DNA, or may be present on the same replicon as the T-DNA, or in trans on a compatible replicon in the Agrobacterium host. Preferably the trans acting factors required for T-DNA transfer are present on the same replicon as the T-DNA.
By "Agrobacterium replicon" is meant any replicon (e.g., plasmid or other vector) that is capable of being stably maintained in an Agrobacterium host. Such replicons include the Agro bacterium chromosome, Agro bacterium plasmids, cosmids, phagemids, etc., derivatives thereof, and any other vector capable of stable replication in Agrobacterium. For example, a binary vector suitable for both Agrobacterium-mediated transfer and for facile recombinant manipulations and replication in other organisms is useful in the methods and compositions of the invention. Preferably the replicon is a Ti or Ri plasmid or a derivative thereof.
In the compositions and methods of the invention, the viral replicon is flanked by directly repeated target sites for a site-specific recombinase. By "flanked by" is meant that the target sites may be directly contiguous with the viral DNA or there may be one or more intervening sequences present between one or both ends of the viral DNA and the target sites. Intervening sequences of particular interest would include linkers, adapters, selectable markers and/or other sites which aid in vector construction or analysis and expression cassette for a gene of interest. Target sites for site-specific recombinases are known to those skilled in the art and are discussed in provisional application 60/065,613, filed on Nov. 18, 1997. Examples of target sites include, but are not limited to FRT, FRT1, FRT5, FRT6, FRT7, other FRT mutants, loxP, loxP mutants, and the like. The loxP and FRT sites are shown in
By "directly repeated" is meant that the target sites that flank the viral DNA are arranged in the same orientation, so that recombination between these sites results in excision, rather than inversion, of the viral DNA.
By "site-specific recombinase" is meant any enzyme capable of being functionally expressed in plants, that catalyzes conservative site-specific recombination between its corresponding target sites. For reviews of site-specific recombinases, see Sauer (1994) Current Opinion in Biotechnology 5:521-527; Sadowski (1993) FASEB 7:760-767; the contents of which are incorporated herein by reference. Methods of using site-specific recombination systems to excise DNA fragments from chromosomal or extrachromosomal plant DNA are known to those skilled in the art. The bacteriophage P1 loxP-Cre and the Saccharomyces 2μ plasmid FRT/FLP site-specific recombinations systems have been extensively studied. For example, Russell et al. (1992, Mol. Gen. Genet. 234:49-59) describe the excision of selectable markers from tobacco and Arabidopsis genomes using the loxP-Cre site-specific recombination system.
It is furthermore recognized that the recombinase, which is used in the invention, will depend upon the target sites of the transformed organism and the targeting cassette. That is, if FRT sites are utilized, the FLP recombinase will be needed. In the same manner, where lox sites are utilized, the Cre recombinase is required. If the non-identical target sites comprise both a FRT and a lox site, both the FLP and Cre recombinase or a chimeric recombinase having both Cre and FLP functions, as described in the concurrently filed provisional application entitled "A NOVEL METHOD FOR THE INTEGRATION OF FOREIGN DNA INTO EUKARYOTIC GENOMES," will be required in the plant cell.
The site-specific recombinase may be a naturally occurring recombinase or an active fragment derivative thereof. Site-specific recombinases useful in the methods and compositions of the invention, include recombinases from the integrase and resolvase families, derivatives thereof, and any other naturally occurring or recombinantly produced enzyme or derivative thereof, that catalyzes conservative site-specific recombination between specified DNA sites. The integrase family of recombinases has over thirty members and includes FLP, Cre, Int and R. The resolvase family includes γδ resolvase. Recombinant enzymes that catalyze site-specific conservative recombination include moFLP, described in application Ser. No. 08/972,258, now U.S. Pat. No. 5,929,301. Other recombinases include moCre described in U.S. patent application Ser. No. 09/193,503, filed Nov. 17, 1998, the contents of which are incorporated herein by reference. Preferably, the recombinase is one that does not require cofactors or a supercoiled substrate. Such recombinases include Cre, FLP moflp, and moCre.
moflp is derived from the Saccharomyces 2μ plasmid FLP recombinase, but is encoded by a nucleic acid sequence utilizing maize-preferred codons. While the moflp nucleic acid sequence includes preferred codons for expression of amino acids in maize, it is understood that a useful sequence may contain codons occurring in maize with less than the highest reported maize codon frequencies.
The site-specific recombinases and sequences encoding them that are used in the methods and compositions of the invention may be variants of naturally occurring recombinases and the genes encoding them. The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for it's native substrate.
Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See Creighton (1984) Proteins, W.H. Freeman and Company.
When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucl. Acids Res. 17:477-498; and Campbell et al. (1990) Plant Physiol. 92:1). Thus, the maize preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants are listed in Table 4 of Murray et al., supra.
The FLP recombinase gene from yeast (Saccharomyces cerevisiae) is commercially available in plasmid pOG44 from Stratagene Cloning Systems (11011 North Torrey Pines Road, La Jolla, Calif. 92037). Similarly, the sequences of many other site specific recombinases and their cognate target sites are publicly or commercially available.
Genes encoding site-specific recombinases, viral DNA, T-DNA and target sites can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art and can be found in such references as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, N.Y., 1989). A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and which choices can be readily made by those of skill in the art. For a description of various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, Ill.). Genes encoding FLP can be obtained, for example, by synthesizing the genes with mutually priming long oligonucleotides. See, for example, Ausubel et al. (eds.), Current Protocols In Molecular Biology, pages 8.2.8 to 8.2.13, Wiley Interscience (1990). Also, see Wosniak et al. (1987) Gene 60:115. Moreover, current techniques using the polymerase chain reaction provide the ability to synthesize genes as large as 1.8 kilobases in length (Adang et al. (1993) Plant Mol. Biol. 21:1131; Bombat et al. (1993) PCR Methods and Applications 2:266).
Rather than use full length recombinases, functional fragments of site-specific recombinases may be used in the methods and compositions of the invention. Functional fragments of site-specific recombinases can be identified using a variety of techniques such as restriction analysis, Southern analysis, primer extension analysis, and DNA sequence analysis. Primer extension analysis or S1 nuclease protection analysis, for example, can be used to localize the putative start site of transcription of the cloned gene. Ausubel at pages 4.8.1 to 4.8.5; Walmsley et al., Quantitative and Qualitative Analysis of Exogenous Gene Expression by the S1 Nuclease Protection Assay, in Methods In Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols, Murray (ed.), pages 271-281 (Humana Press, Inc. 1991). For examples, functional fragments of the FLP protein may be identified by their ability, upon introduction to cells containing appropriate FRT substrates, to catalyze site-specific recombination (for example, excision of an FRT-flanked sequence which upon removal will activate an assayable marker gene.
The general approach of such functional analysis involves subcloning DNA fragments of a genomic clone, cDNA clone or synthesized gene sequence into an expression vector, introducing the expression vector into a heterologous host, and screening to detect the product of recombination (i.e. using restriction analysis to verify the product of recombination at the nucleic acid level, or relying on an assay system for recombination as described above). Methods for generating fragments of a cDNA or genomic clone are well known. Variants of an isolated DNA encoding a site-specific recombinase can be produced by deleting, adding and/or substituting nucleotides. Such variants can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like. See, for example, Ausubel, pages 8.0.3-8.5.9. Also, see generally, McPherson (ed.), Directed Mutagenesis: A Practical approach, (IRL Press, 1991).
The nucleic acid sequence encoding the site-specific recombinase is operably linked to a promoter that drives expression in a plant. As used herein "operably linked" includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria genes that are expressed in plant cells such as those of Agrobacterium or Rhizobium. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of a sequence encoding a site-specific recombinase. The promoter may be constitutive, inducible or tissue specific.
Many different constitutive promoters can be utilized in the instant invention. Exemplary constitutive promoters include the promoters from plant viruses such as the 35S promoter from CaMV (Odell et al. (1985) Nature 313:810-812) and the promoters from such gene as rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); maize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. 231: 276-285 and Atanassova et al. (1992) Plant Journal 2(3):291-300); the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the Pemu promoter, the rubisco promoter, the GRP 1-8 promoter, and other transcription initiation regions from various plant genes known to those of skill. The ALS promoter, a XbaI/NcoI fragment 5-prime to the Brassica napus ALS3 structural gene (or a nucleotide sequence that has substantial sequence similarity to said XbaI/NcoI fragment), represents a particularly useful constitutive promoter for dicots. (See Pioneer Hi-Bred International U.S. patent application Ser. No. 08/409,297, now U.S. Pat. No. 5,659,026).
A variety of inducible promoters can be used in the instant invention. See Ward et al. (1993) Plant Mol. Biol. 22:361-366. Exemplary inducible promoters include that from the ACE1 system which responds to copper (Mett et al. (1993) PNAS 90:4567-4571); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (Hershey et al. (1991) Mol. Gen. Genetics 227:229-237 and Gatz et al. (1994) Mol. Gen. Genetics 243:32-38); the Adh1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, and the PPDK promoter which is inducible by light; or Tet repressor from Tn10 (Gatz et al. (1991) Mol. Gen. Genet. 227:229-237. A particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:10421).
Examples of promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
The site-specific recombinase must be expressed in the plant cell in order for mobilization of the viral DNA from the T-DNA. Accordingly, the recombinase protein must be expressed so that it is present in the cell at an effective concentration sometime between when the T-DNA is transferred to the plant cell and before non-integrated T-DNA is lost from the plant cells.
Accordingly, the expression cassette encoding the site-specific recombinase may be supplied on the T-DNA in cis to the viral DNA; in trans on a plant chromosome or extrachromosomal replicon; or may be transferred to the plant near to the time of agro-mediated transformation. The site-specific recombinase gene may be constitutively or transiently expressed, or may be inducible. If cofactors are required for a particular site-specific recombination system, they may be encoded on the T-DNA of the invention or in the plant host chromosomal or extrachromosomal DNA.
By "target site for a site-specific recombinase" is meant a DNA sequence that is recognized by a particular site-specific recombinase. A variety of target sites are known to those skilled in the art and may be used in the methods and compositions of the invention. The site may have the sequence of the cognate site for a given recombinase, or may be modified, so long as it is capable of acting as a target site. The site may be contain the minimal sequences necessary for recombination, or it may contain additional sequences that enhance recombination. Examples of target sites for use in the invention are known in the art and include FRT and loxP sites (See, for example, Schlake and Bode (1994) Biochemistry 33:12746-12751; Huang et al. (1991) Nucleic Acids Research 19:443-448; Paul D. Sadowski (1995) In Progress in Nucleic Acid Research and Molecular Biology, Vol. 51, pp. 53-91; Michael M. Cox (1989) In Mobile DNA, Berg and Howe (eds) American Society of Microbiology, Washington D.C., pp. 116-670; Dixon et al. (1995) 18:449-458; Umlauf and Cox (1988) The EMBO Journal 7:1845-1852; Buchholz et al. (1996) Nucleic Acids Research 24:3118-3119; Kilby et al. (1993) Trends Genet. 9:413-421: Rossant and Geagy (1995) Nat. Med. 1: 592-594; Lox Albert et al. (1995) The Plant J. 7:649-659: Bayley et al. (1992) Plant Mol. Biol. 18:353-361; Odell et al. (1990) Mol. Gen. Genet. 223:369-378; and Dale and Ow (1991) Proc. Natl. Acad. Sci. USA 88:10558-105620; Qui et al. (1994) Proc. Natl. Acad. Sci. USA 91:1706-1710; Stuurman et al. (1996) Plant Mol. Biol. 32:901-913; Hartley et al. (1980) Nature 286: 860-864; Sauer (1994) Current Opinion in Biotechnology 5:521-527; and Dale et al. (1990) Gene 91:79-85; all of which are herein incorporated by reference.)
Each loxP and FRT site contains two 13 base pair inverted repeats which flank an 8 base pair spacer. The FRT site contains an additional non-essential 13 base pair repeat. The sequences of the loxP and FRT sites are shown in
T-DNAs containing viral DNA flanked by recombinations sites, expression cassettes for site-specific recombinases and vectors carrying these sequences may be constructed using standard molecular biology techniques. See, for example, Sambrook et al. (eds.) Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. 1989).
Typical vectors useful in the methods and compositions of the invention are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al, (1987) Meth. in Enzymol., 153:253-277. These vectors are plant integrating vectors in that on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant. Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc. Natl. Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein is plasmid pBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto, Calif.).
Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising et al., Ann. Rev. Genet. 22: 421-477 (1988). These methods are useful for transforming a plant cell with a site-specific recombinase expression cassette. This step will be necessary if the cassette is not included in the transferred T-DNA. The expression cassette encoding the site-specific recombinase may be present in the plant genome prior to Agroinfection or may be transformed into the plant around the time of transfer of the T-DNA to the plant cell so that it will be transiently expressed. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, PEG poration, particle bombardment, silicon fiber delivery, microinjection of plant cell protoplasts or embryogenic callus, or Agrobacterium-mediated transformation (Hiei et al. (1994) Plant J. 6:271-282).
The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al., Embo J. 3: 2717-2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Natl. Acad. Sci. 82: 5824 (1985). Ballistic transformation techniques are described in Klein et al., Nature 327: 70-73 (1987).
DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al., Methods in Enzymology, 101:433 (1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., Plant Mol. Biol. Reporter, 6:165 (1988). Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al., Nature, 325:274 (1987). DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus et al., Theor. Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
Plants cells stably transformed with a site-specific recombinase expression cassette can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of plant Cell Culture, MacMillan Publishing Company, New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).
The regeneration of plants containing the recombinase gene introduced by Agrobacterium from leaf explants can be achieved as described by Horsch et al., Science, 227:1229-1231 (1985). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.
Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis. (1988).
One of skill will recognize that after the site-specific recombinase expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
The methods and compositions of the invention are useful to mobilize viral DNA from T-DNAs transferred by agroinfection to any plant host. As used herein, the term "plant" includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. Plant cell, as used herein includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to Agrobacterium transformation techniques, including both monocotyledonous and dicotyledonous plants. A particularly preferred monocot is maize. Other monocots of particular interest include wheat, rice, barley, sorghum and rye. Dicots of particular interest include soybean, Brassica, sunflower, alfalfa, and safflower.
The T-DNA containing the viral DNA flanked by sites for a site-specific recombinase is transferred to a plant cell by agroinfection. Agrobacterium tumefaciens-meditated transformation techniques are well described in the scientific literature. See, for example Horsch et al. Science 233: 496-498 (1984), Fraley et al. Proc. Natl. Acad. Sci. 80: 4803 (1983); and Kado (1991) Crit. Rev. Plant Sci. 10:1. Descriptions of the Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provide in Gruber et al., supra; Miki, et al., supra; and Moloney et al. (1989) Plant Cell Reports 8:238. Although Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium. For instance, Agrobacterium transformation of maize is described in U.S. Pat. No. 5,550,318. Other methods of agroinfection include Agrobacterium rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, Vol. 6, P W J Rigby, Ed., London, Academic Press, 1987; and Lichtenstein, C. P., and Draper, J., In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985), Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use of A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16.
Optimized methods and vectors for Agrobacterium-mediated transformation of plants in the family Graminae, such as rice and maize have been described by Heath et al. (1997) Mol. Plant-Microbe Interact. 10:221-227; Hiei et al. (1994) Plant J. 6:271-282 and Ishida et al. (1996) Nat. Biotech. 14:745-750, the contents of which are incorporated herein by reference. The efficiency of maize transformation is affected by a variety of factors including the types and stages of tissue infected, the concentration of Agrobacterium, the tissue culture media, the Ti vectors and the maize genotype. Super binary vectors carrying the vir genes of Agrobacterium strains A281 and A348 are useful for high efficiency transformation of monocots. However, even without the use of high efficiency vectors, it has been demonstrated that T-DNA is transferred to maize at an efficiency that results in systemic infection by viruses introduced by agroinfection, although tumors are not formed (Grimsley et al. (1989) Mol. Gen. Genet. 217:309-316, the contents of which are incorporated herein by reference). This is because integration of the T-DNA containing the viral genome is not required for viral multiplication, since the excised viral genome acts as an independent replicon.
Another useful basic transformation protocol involves a combination of wounding by particle bombardment, followed by use of Agrobacterium for DNA delivery, as described by Bidney et al. (1992) Plant Mol. Biol. 18:301-313. In general, the intact meristem transformation method involves imbibing seed for 24 hours in the dark, removing the cotyledons and root radical, followed by culturing of the meristem explants. Twenty-four hours later, the primary leaves are removed to expose the apical meristem. The explants are placed apical dome side up and bombarded, e.g., twice with particles, followed by co-cultivation with Agrobacterium. To start the co-cultivation for intact meristems, Agrobacterium is placed on the meristem. After about a 3-day co-cultivation period the meristems are transferred to culture medium with selection agents.
The split meristem method involves imbibing seed, breaking of the cotyledons to produce a clean fracture at the plane of the embryonic axis, excising the root tip and then bisecting the explants longitudinally between the primordial leaves. The two halves are placed cut surface up on the medium then bombarded twice with particles, followed by co-cultivation with Agrobacterium. For split meristems, after bombardment, the meristems are placed in an Agrobacterium suspension for 30 minutes. They are then removed from the suspension onto solid culture medium for three day co-cultivation. After this period, the meristems are transferred to fresh medium with cefotaxime (plus selection agents).
The excision of viral DNA from the T-DNA may be determined by a variety of means including but not limited to observation of viral lesions or other signs of viral infection on the plant, by antibody-based detection of viral proteins, by amplification of PCR products specific to a circular genome, or by Northern analysis for viral RNA. In addition, assays for FLP recombinase activity are known and generally measure the overall activity of the enzyme on DNA substrates containing FRT sites. In this manner, a frequency of excision of the sequence can be determined. Excision of DNA from a linear molecule or intermolecular recombination frequency induced by the enzyme may be assayed, as described, for example, in Babineau et al. (1985) JBC 260:12313; Meyer-Leon et al. (1987) NA Res 15:6469; and Gronostajski et al. (1985) JBC 260:12328.
The following examples are offered by way of illustration not by way of limitation.
DNA fragments containing viral DNA flanked by FRT or loxP site-specific recombination sites are constructed either by synthesizing, annealing and ligating complementary oligonucleotides or by creating primers for PCR amplification of a DNA product containing the FRT or loxP site and restriction sites useful for cloning into T-DNA near the 5′ end of the PCR product.
For example, long PCR primers may be designed wherein the 3′ end of the primer hybridizes to the 5′ end of the viral genome of interest and the 5′ end of the primer further contains a loxP site and useful cloning sites (see
Nicotiana tabacum BY-2 cells are propagated in Murashige and Skoog medium (Gibco BRL) containing 3% sucrose, 1 μg/ml thiamine, 0.2 μg/ml 2,4-D, and 370 μg/ml KH2PO4 . Zea mays Black Mexican Sweet (BMS) cells are propagated in Murashige and Skoog medium containing 2% sucrose, 2 μg/ml 2,4-D, 0.2 mg/ml myoinositol, 0.13 mg/ml L-asparagine, 0.13 μg/ml nicotinic acid, and 0.25 μg/ml each of thiamine, pyridoxine, and pantothenic acid. The cultures are shaken at 140 rpm at 25� in continuous light.
To infect plant cells, virulence (vir) gene activity is induced by treatment with acetosyringone in Agrobacterium carrying the vector constructed in Example 1. Agrobacterium cells are grown to a density of 2�109 cells per ml (A=100, using a Klett-Summerson spectrophotometer, red filter) in AB-sucrose medium. The cells are centrifuged at 10,000 g, suspended at a concentration of 1�109 cells per ml (A=50) in induction medium (AB salts, 0.5% glucose, 2 mM sodium phosphate, 50 Mm Mes, Ph 5.6, 50 μM acetosyringone), and incubated with gentle shaking at 25� for 14 to 18 hr. After washing the bacterial cells in plant culture medium, plant cells are inoculated with induced Agrobacterium (−20 bacterial cells per plant cell, except where noted otherwise) and cocultivated at 25� with shaking at 140 rpm for various periods of time. Most of the bacteria is washed off by centrifugation of the cocultivation mixture at 300 rpm (model GLC-2 clinical centrifuge; Beckman Sorvall, Newtown, Conn.) for 2 min. The plant cell pellet is suspended and washed once more in plant culture medium and then resuspended in culture containing either 100 μg/ml timentin or 200 μg/mL cefotaxime. Mobilization of viral genomes from the T-DNA is monitored by quantitating infectious viral particles.
Prior to inoculation, strains of Agrobacterium carrying the vector constructed in Example 1 are streaked out on YEB (Grimsley et al. (1986) Proc. Natl. Acad. Sci. USA, 83:3282-3286) plates solidified with 1.5% agar and supplemented with 100 μg/ml rifampicin and 25 μg/ml kanamycin and allowed to grow for 48 h. A single colony is used to inoculate 10 ml of liquid YEB medium in a 100 ml Erlenmeyer flask supplemented with antibiotics as previously. Growth is continued with shaking at 200 r.p.m. for 24 h, then 500 μl of this culture is used to inoculate a similar flask and growth continued for a further 20 h. This procedure yields a final density of viable Agrobacterium cells in the region of 109/ml (estimated by plating). The cells are then harvested by centrifugation and resuspended in an equal volume of 10 mM MgSO4 without antibiotics; such a suspension is subsequently referred to as undiluted or 100 dilution; for experiments involving a dilution series 10 mM MgSO4 was also used as the diluent.
Maize seeds for 10-day old plants are sown in pots in a phytotron in a 12 hour light/dark cycle at 25� in a light intensity of about 10000 lux (Sylvania 215 W fluorescent lamps type F96T12/CW/VHO) then moved to the BL3 containment laboratory immediately prior to inoculation; subsequent growth conditions have been described (Grimsley et al. (1987) Nature 325:177-179). Three-day old seedlings are prepared by (i) sterilization by stirring for 20 min in 0.7% calcium hypochlorite solution, (ii) washing three times (stirring for 20 min each time) in sterile distilled water (iii) preparing 9 cm diameter presterilized Petri dishes with 3 sheets of sterile 8.5 cm diameter Macherey-Nagel (Germany) filter paper in the bottom and ca. 10 ml of sterile water per dish, (iv) putting ca. 20 seeds into each geranium dish, and (v) incubating in the dark at 28� for 3 days, or until the distance between the scutellar node and the apical tip of the coleoptiles is 1-2 cm.
For inoculation of plants, a 50 μl or a 100 μl Hamilton syringe fitted with a 0.4 mm diameter disposable needle is loaded with the bacterial suspension avoiding trapped air bubbles. Between inoculations with different bacterial strains the needle is discarded and the syringe flushed out 3 times with 100% ethanol and 3 times with sterile distilled water. 10-day old plants are inoculated by (i) abrasion of an upper leaf, applying 20 μl of suspension, and rubbing in with carborundum powder until the leaf appears wet all over, (ii) injection of 10 μl of bacterial suspension into the central part of the plant either just above the first leaf blade, or 1 cm below the first leaf blade, or at the base of the plant, in the meristematic region where adventitious roots later begin to appear. Three-day old seedlings are injected with 10 μl of bacterial suspension in different ways by (i) pushing the needle down through the apical tip of the coleoptile to the coleoptilar node, (ii) injecting 2 mm below the apical tip of the coleoptile, (iii) 2 mm above the coleoptilar node, (iv) at the coleoptilar node, (iv) 2 mm below the coleoptilar node, (v) at the scutellar node, and by pushing the needle up through the primary root to a region close to the scutellar node. Ten μl is used as a standard inoculum of bacterial suspension, but only 1-2 μl routinely remains in the inoculation site, the rest is forced out, usually coming out from the point of entry of the inoculating needle. Following inoculation seedlings are planted immediately in damp soil, incubated as before (Grimsley et al. (1987) Nature 325:177-179), and observed daily for the appearance of symptoms of viral infection, characterized by the appearance of yellow spots and/or stripes at the base of new leaves.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
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|Publication date
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|Oct 14, 1997
|The Salk Institute For Biological Studies
|FLP-mediated gene modification in mammalian cells, and compositions and cells useful therefor
|Mar 8, 1996
|Apr 28, 1998
|Purdue Research Foundation
|DNA constructs for controlled transformation of eukaryotic cells
|May 29, 1996
|Jan 4, 2000
|Medical Research Council
|Recombinant binding proteins and peptides
|Jan 12, 1998
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|Life Technologies, Inc.
|Recombinational cloning using engineered recombination sites
|Nov 17, 1998
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|Pioneer Hi-Bred International, Inc.
|Compositions and methods for genetic modification of plants
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|Jul 17, 2001
|Pioneer Hi-Bred International, Inc.
|Method for the integration of foreign DNA into eukaryotic genomes
|Nov 17, 1998
|Oct 9, 2001
|Pioneer Hi-Bred International, Inc.
|Mobilization of viral genomes from T-DNA using site-specific recombination systems
|Nov 17, 1998
|Dec 18, 2001
|Pioneer Hi-Bred International, Inc.
|Method for directional stable transformation of eukaryotic cells
|Nov 12, 1999
|Sep 24, 2002
|Pioneer Hi-Bred International, Inc.
|Compositions and methods to stack multiple nucleotide sequences of interest in the genome of a plant
|Nov 12, 1999
|Oct 1, 2002
|Pioneer Hi-Bred International, Inc.
|Compositions and methods for the targeted removal of a nucleotide sequence from the genome of a plant
|Oct 12, 1999
|Apr 1, 2003
|Pioneer Hi-Bred International, Inc.
|Method for the integration of foreign DNA into eukaryotic genomes
|Nov 12, 1999
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|Pioneer Hi-Bred International, Inc.
|Compositions and methods for locating preferred integration sites within a plant genome
|Nov 12, 1999
|Jun 3, 2003
|Pioneer Hi-Bred International, Inc.
|Compositions and methods to reduce the complexity of transgene integration in the genome of a plant
|Dec 6, 1999
|Sep 23, 2003
|Pioneer Hi-Bred International, Inc.
|Compositions for the genetic modification of plants
|May 30, 1997
|Aug 10, 2004
|Carnegie Institution Of Washington
|Use of FLP recombinase in mice
|Jan 29, 2003
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|Pioneer Hi-Bred International, Inc.
|Novel method for the integration of foreign DNA into eukaryotic genomes
|Title not available
|Mar 6, 1992
|Sep 17, 1992
|The Salk Institute For Biological Studies
|Flp-mediated gene modification in mammalian cells, and compositions and cells useful therefor
|Jul 6, 1992
|Jan 21, 1993
|The United States Of America As Represented By The Secretary Of Agriculture
|Selection-gene-free transgenic plants
|Feb 25, 1993
|Sep 2, 1993
|Mogen International N.V.
|Agrobacterium strains capable of site-specific recombination
|Jan 27, 1994
|Aug 4, 1994
|Purdue Research Foundation
|Controlled modification of eukaryotic genomes
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|European Molecular Biology Laboratory (Embl)
|Regulation of site-specific recombination by site-specific recombinase/nuclear receptor fusion proteins
|Dec 5, 1994
|Jun 8, 1995
|Medical Research Council
|Recombinant binding proteins and peptides
|Jul 31, 1995
|Feb 15, 1996
|Delta And Pine Land Company
|Control of plant gene expression
|Sep 5, 1996
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|Crc For Biopharmaceutical Research Pty. Ltd.
|Method for producing phage display vectors
|Aug 27, 1996
|Mar 13, 1997
|Genvec, Inc.
|Methods and vectors for site-specific recombination
|Oct 11, 1996
|Apr 17, 1997
|Purdue Research Foundation
|Method for the production of hybrid plants
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|Commonwealth Scientific And Industrial Research Organisation
|Single-step excision means
|Jun 6, 1997
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|Massachusetts Institute Of Technology
|Methods and compositions for transforming cells
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|May 14, 1999
|The United States Of America, As Represented By The Secretary Of Agriculture
|Resolution of complex integration patterns to obtain single copy transgenes
|Apr 26, 1999
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|Novartis Ag
|Site-directed transformation of plants
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|Lyznik et al., Activity of Yeast FLP Recombinase in Maize and Rice Protoplasts, Nucleic Acids Research, 1993, pp. 969-975, vol. 21. No. 4, Oxford University Press.
|Lyznik et al., FLP-Mediated Recombination of FRT Sites in the Maize Genome, Nucleic Acids Research, 1996, pp. 3784-3789, vol. 24, No. 19, Oxford University Press.
|McLeod et al. Identification of the Crossover Site During FLP-Mediated Recombination in the Saccharomyces cerevisiae Plasmid 2mum Circle, Mol. Cell. Biol., Oct. 1986, pp. 3357-3367, vol. 6(10), American Society for Microbiology, Cold Spring Harbor, New York.
|McLeod et al. Identification of the Crossover Site During FLP-Mediated Recombination in the Saccharomyces cerevisiae Plasmid 2μm Circle, Mol. Cell. Biol., Oct. 1986, pp. 3357-3367, vol. 6(10), American Society for Microbiology, Cold Spring Harbor, New York.
|Murray, et al., "Condon Usage in Plant Genes," Nucleic Acid Research, 1989, pp. 477-490, vol. 17(2).
|Narasimhulu et al., Early Transcription of Agrobacterium T-DNA Genes in Tobacco and Maize, The Plant Cell, May 1996, pp. 873-886, vol. 8, American Society of Plant Physiologists.
|O'Gorman et al. Recombinase-Mediated Gene Activation and Site-Specific Integration in Mammalian Cells, Science, Mar. 15, 1991, pp. 1351-1355, vol. 251, Salk Institute for Biological Studies, La Jolla, California.
|O'Gorman et al., Protamine-Cre Recombinase Transgenes Efficiently Recombine Target Sequences in the Male Germ Line of Mice, but not in Embryonic Stem Cells, Proc. Natl. Acad. Sci. USA (Dec. 1997) pp. 14602-14607 vol. 94 Gene Expression Lab., The Salk Inst. For Biological Studies, San Diego, CA.
|Osborne, B.I., et al., "A System for Insertional Mutagenesis and Chromosomal Rearrangement Using the Ds Transposon and Cre-lox," The Plant Journal, 1995, pp. 687-701, vol. 7(4).
|Ow et al., Genome Manipulation Through Site-Specific Recombination, Critical Reviews in Plant Sciences, (1995), pp. 239-261, vol. 14(3).
|Raineri et al., "VirA, The Plant-Signal Receptor, is Responsible for the T1 Plasmid-specific Transfer of DNA to Maize by Agrobacterium," Microbiology (1993) pp. 3549-3553, vol. 90.
|Russell, et al., Directed Escision of a Transgene from the Plant Genome, Mol. Genet Genet (1992), pp. 49-59, vol. 234 Central Res. and Devel. And Agric. Prod. Du Pont Experimental Station, Wilmington, DE.
|Sadowski et al., "Site Specific Genetic Recombination: Hops, Flips, and Flops," FASEB, 1993, pp. 760-767, vol. 7.
|Sauer (1992) "Identification of Cryptic lox Sites in the Yeast Genome by Selection for Cre-mediated Chromosome Translocations that Confer Multiple Drug Resistance", J. Mol. Biol. 223:911-928.
|Sauer, B., "Site-Specific Recombination: Developments and Applications," Current Opinion in Biotechnology, 1994, pp. 521-527, vol. 5.
|Schlake et al,. "Use of Mutated FLP Recognition Target (FRT) Sites for the Exchange of Expression Cassettes at Defined Chromosomal Loci," Biochemistry (1994) pp. 12746-12751 vol. 33:GBF, Braunschweig-Stockheim, Germany.
|Scholthof et al. (1996) "Plant Virus Gene Vectors For Transient Expression of Foreign Proteins in Plants", Annu. Rev. of Phytopathohl 34:299-323.
|Seibler et al. (1997) "Double-Reciprocal Crossover Mediated by FLP-Recombinase: A Concept and an Assay", Biochemistry 36;1740-1747.
|Senecoff et al. (1988) "DNA Recognition By The FLP Recombinase of the Yeast 2 mu Plasmid", J. Mol. Biol. 201:406-421.
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|Senecoff et al. Directionality in FLP Protein-Promoted Site-Specific Recombination Is Mediated by DNA-DNA Pairing, J. Biol. Chem., Jun. 5, 1986, pp. 7380-7386, vol. 261(16), The American Society of Biological Chemists, Inc., Madison, Wisconsin.
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|Timmermans, et al., "Geminiviruses and Their Uses as Extrachromosomal Replicons," Annu. Rev. Plant Physiol. Plant Mol. Biol., 1994, pp. 79-112, vol. 45.
|U.S. Appl. No. 09/455,051, filed Dec. 6, 1999, Baszczynski et al.
|U.S. Appl. No. 10/430,907, filed May 7, 2003, Baszczynski et al.
|U.S. Appl. No. 10/430,908, filed May 7, 2003, Baszczynski et al.
|U.S. Appl. No. 10/440,030, filed May 16, 2003, Baszczynski et al.
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|Ugaki et al. (1991) "Replication of a Geminivirus Derived Shuttle Vector in Maize Endosperm Cells", Nucleic Acids Research 19(2):371-377.
|Umlauf et al,."The Functional Significance of DNA Sequence Structure in a Site-Specific Genetic Recombination Reaction," 1988, pp. 1845-1852, IRL Press Limited, Oxford, England.
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|U.S. Classification
|800/278, 800/293
|International Classification
|Cooperative Classification
|European Classification


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Owen Wilson: I was starstruck by Pierce Brosnan
Hollywood star Owen Wilson admitted he was a bit star struck when Pierce Brosnan arrived on the set of their new movie.
The pair are currently starring in thriller No Escape opposite Lake Bell.
The movie tells the story of a businessman and his family attempting to escape a foreign country in the midst of a coup.
"When he came on set in Chiang Mai in Thailand it was exciting because you can't help but think 'James Bond is walking around'," he said.
"Even in New York, I was walking down the street with Pierce and we went to get something to eat and seeing the reaction that people have is incredible.
"When you play James Bond you occupy a space in people's consciousness and it's funny to see how people react," he added to Anton Savage on Today FM.
While the Navan man has lots of fans Stateside, he was also incredibly popular in Thailand.
"I did have a little bit of a Thai following, but when Pierce showed up, it was nothing compared to that," Owen said.
Despite some negative reviews, the thriller, which had a budget of just €4.5m, did well at the US Box Office last weekend, pulling in €7.4m.
While his star status is thanks to his 007 role, Pierce recently admitted he was in line to play Batman in Tim Burton's 1989 movie but turned down the role because he was "naive".
"I was kind of hesitant about the whole thing. I couldn't quite get my head around it," Brosnan said.
"I knew it was Batman but I just didn't think that was going to fly. I was silly."
The 62-year-old will next be on the silver screen in thriller I.T which he shot in Dublin.

subset: commoncrawl


text:
Gallery of Pixie Lott bikini candids in Mauritius
Taking a plunge at the swimming pool is a great feeling and bringing the heat to the cool water is hot Latina Karina, who shows off wearing a yellow bikini and strips it off. Teasing in front of the guy, he just couldn't believe at what he's seeing as she does her horny plays with him. She goes to the pool and let him lick her sweet pussy then spreads her legs to invite his cock to enter inside. She's gets a hard pounding doggy fuck she's craving for and gets mouthful load.
Sofia Vergara loosens up and drops her bikini revealing cleavage
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Jessica Alba nonnude wearing a bikini in her pre-pregnancy days
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Leanne unzips her bikini in the pool
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Sexy bombshell Jennifer Nicole Lee's bikini malfunction
Jennifer Lawrence's pumped teen boobs get wet with water
Stunning babe in bikini licks her fingers before thrusting it on her pussy and cute perky tits
Hot sexy Michelle Rodriguez gets wet in a skimpy bikini
Curvy Frankie Essex busty in a sexy red bikini
Vanessa Hudgens nonnude in a red bikini and loose sexy top
Joanna Krupa sexy bikini carwash candid photos
Curvy Thai in bikini
Hot Apriloneil in her sexy red bikini and teasing as she strips and gets wet in the pool
Birthday slut Miley Cyrus' selfshooting and bikini pictures
Teen jenni on cam takes off her bikini tops showing lickable yummy tits
Hottie teen in mini skirt Maddie showing off her bikini and her tiny pink pussy
Cameron Diaz nonnude and hosing herself wet while in a blue bikini
Maria Kanellis shows her curvy body dressed in bikini in pictures
Collection of sexy Johanna Lundback Nelly bikini photoshoot
Lorena Bitch is one sexy bitch. Her body is what you wanna see in a thin bikini or barely there underwear. We know what you're thinking.
Gorgeous Whitney Port flaunts her bikini body in Miami
Ultra sexy blonde seductress Chloe teasing us with her pink pantyhose and black bikini
Leanne in the pool with a zip-up bikini
Adrianna and Destiny - DildoFucking Duo - Two bikini babes fuck with a dildo
Victoria Silvstedt skimpy bikini candids in St. Barts


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Words that start with blackac
Found 3 words that start with blackac. Browse our Scrabble Word Finder, Words With Friends cheat dictionary, and WordHub word solver to find words starting with blackac. Or use our Unscramble word solver to find your best possible play! Related: Words containing blackac
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Home Details
Listing Details
Property Virtual Tour
This Pupukea treasure has stunning ocean and sunset views. The heavenly blend of dramatic architecture, lush landscaping and distinctive interior design defines this Pupukea property as a premier estate on the North Shore of Oahu.
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Enjoy the feeling of your very own private retreat!
Home Details
Location and Neighborhood
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- Property Location
Real Estate Virtual Tour and Photography by PanaViz


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Item description for How To Live Worry Free by Kenneth Hagin, Jr....Overview
Is it possible to live completely worry-free?
God has called us as believers to live in peace, free from the entanglements of worry, fear, and anxiety that hold many in bondage today.
There is a solution to the tests and trials of life and the everyday stress of trying to "keep your head above water" and solving life's problems in your own strength. The answer is found in relying upon God's love, care, and concern for you. His infinite wisdom and power are able to put you over and make you a success in every area of your life. You don't have to worry, because God cares for you!
In How To Live Worry-Free, Rev. Kenneth Hagin Jr. combines sound teaching from God's Word with practical insights to illustrate the perils of worry and to help guide the believer into God's safe haven of peace that passes all understanding. Rev. Hagin Jr. teaches that by practically applying God's Word to life's problems, you can live free from the bondage of worry and allow God to work in your life to turn around negative situations and circumstances.
It is possible for you to live comletely worry-free!
In this book you will also discover:
-Can You Be Worry Free?
-The Destructive Power Of Worry
-You Have Authority Over Worry!
Publishers Description
Sound teaching from God's Word is combined with practical insights in this book that deals with the perils of worry and provides a guide for the believer into God's safe haven of peace.
Promise Angels is dedicated to bringing you great books at great prices. Whether you read for entertainment, to learn, or for literacy - you will find what you want at promiseangels.com!
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How to Pack for a Safari
Traveling is a thrilling experience. However, dragging heavy baggage through the airport can be a real pain. Many travelers express frustration regarding what and how much to pack. This is entirely understandable. However, there should be less emphasis on what to bring, and more emphasis on being—fully and presently—at your destination. After months of preparation […]
The Virgin Islands: Caribbean Cruisin'
Ed. note: This is the first part in a series by Women's Adventure contributor Alyssandra Barnes, as she makes her way around the U.S. Virgin Islands and shares her travel tips. And yes, it's snowing right now, so we are a little jealous. First Stop: St. Croix Most people, myself included, only have a vague […]
The Day Travel Changed Me
When traveling abroad, you have to prepare for the unexpected. Here's how to not let it ruin your trip.
The Gilis of Indonesia: Which One Is Right for You?
You could go to Bali. But if you'd prefer an Indonesian island that's more off the beaten track, explore the Gilis.
New Zealand: Adventure Capital of the World
Whether you're bungy jumping, rapelling, sledging, or heli-rafting, there's an adrenaline rush perfect for you in New Zealand.
Urban Adventures: San Francisco
At 7×7 miles, San Francisco is small but teeming with natural beauty and adventure. Here's our outdoorsy guide to the Bay Area.
The End of "The Way": Traditions That Never End
In the final installment of a four-part series on biking the Camino de Santiago, our pilgrim, Gigi Ragland, discusses her arrival at the journey's end: Santiago de Compostela.
Sweet Ski Season Deals and Cyber Monday Specials
Take advantage of some of the great ski lodging deals for the 2013-14 season!
Hotel Homebase: Jackson Hole
This Wild West town offers gorgeous scenery, amazing skiing, and wildlife viewing. We give you the lowdown on how to do Jackson Hole right.
How to Choose an Awesome Guide
A great guide can make or break your trip. Adventurer-in-Residence, Lauren Schaad provides some great tips for finding your perfect match.
A "Cycling Pilgrim's" Journey along the Camino de Santiago: Part 3
In Part 3 of a four-part series, travel editor, Gigi Ragland, discusses the highs and lows of biking the Camino.
Year-round Adventure: How to Live in Your RV all the Time
Ready to make the open road your home? If you've ever thought about traveling full time in your RV, we've got some tips to help make the transition as smooth as possible.


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Chandigarh: Defending champions Bengal defeated Pondichery 3-0 in the girls section of the national sub-junior table tennis championship at Sirsa Wednesday. Meanwhile, in the cadet boys section, Bengal trounced Mizoram and Uttaranchal 3-0.
In the sub-junior boys category, Orissa beat Jammu Kashmir 3-1 while Punjab downed Manipur 3-2.
The defending champions (sub-junior boys) Punjab Sports Control Board (PSCB) outplayed Chandigarh 3-0 while hosts Haryana thrashed Tripura by an identical margin. Jharkhand also went down tamely, losing to Delhi and Chandigarh 0-3.


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Separate names with a comma.
Posted By Shadow&Flame,
Aug 5, 2013 at 12:29 AM
Never saw a stove put together like this one....
I like it! This looks like a nicely developed masonry stove from Poland.The Europeans have these stoves down to a science.
Can't wait until they are common place here. That is awesome, Shadow.
That is so cool...Is this stove working without a chimney???
Better be sure you put it where you want it. I wonder what sort of weight we're talking about here?
Oh... those silly Europeans.
"Don't worry Dear. I am just going to mix some mortar here on the new hardwood floor. What could go wrong?"
A bit more than a ton.
It's vented low and out the back. You can see the flue connection going in around 1:25.
"Finished stove looks good Adelbert. But what is this? I just found it on the porch."
I was I thinking the same thing. If ever there was a time to use nice clean drop clothes it's while shooting a how to video.
Let me get this straight. You order a stove and they drop off a pallet of bricks. One of the options is the two guys in coveralls to help assemble the pile of bricks into something that will not burn the house down?
On a serious note, how airtight is it? Any efficiency numbers? Particulates?
Very interesting. I had to find the assembly guide, and watch the video again to see how it works
On the right/left sides, there are 4 large tubes in the wall of the stove. The exhaust goes to the top of the stove, then back down those tubes to the chimney at the bottom.
The air intake is at the very bottom, then travels up two smaller tubes to the firebox.
I posted a link to the company above. Actually, it's pretty efficient. http://www.termokaust.com/stove/Tests-and-certificates,4.html, http://www.termokaust.com/stove/Technical-specification,2.html
I'm assuming this is not a DIY stove, but may be it is. Personally, I would want a mason to install. I am too sloppy with brick laying. The finished units have some nice tile options.
Pretty cool stove, any idea on why the round piece for the bottom of the firebox, is that a wear item or just decoration?
Yep. Found it in the FAQ.
The floor of the furnace features a plug made of cast iron. It is mounted on a heat-insulating cord, which can be prised off and removed. After opening, we gain access to the sedimentation chamber and outlets of all four sedimentation passes from the top of the stove. Additionally, we have access to the hole of a pipe that connects the stove with chimney. Cleaning consists in periodical removal of the soot after prior clearing all the passes with a wire brush. Access to the top section of the stove is possible through a vault in the furnace.
Thought this was cute:
Remember: burning green wood is disadvantageous to the device! Reasons for using green wood vary. They are innocent sometimes - here are some quotes: "... I liked the smell of burning green wood..."; and our favourite: "...I've been chopping that firewood all day long, why not burn it?...".
Natural inclinations and the enormous desire to burn something eventually win over logical thinking and ecology.
We build firewood stoves taking care of our customer's wallets. Green wood dirty our stove and chimney, and at the same time it produces 50% less energy. Please keep that in mind while using our stoves.
Signed Dionizy (Dennis in Polish)
Sorry, my Polish is not that strong...
With out Union brick layers it may not go over so well here!
These were the first questions I had when I watched the vid.
Hearth and stove all in one... You better make sure you put it where you want it and
it has a sturdy foundation.
Nothing like cooking in the living room.
I saw a video similar to this one a while back but it was a German mini soapstone heater that was put together without any mortar. They used some kind of metal splines and gasket between the joints and she was ready to burn in about an hour. Pretty cool stuff but I don't know if this would fly in the USA.
Knowing german bureaucracy and certification standards I am pretty sure if its ok there it would be ok here. Just as an example: The chimney sweep comes for a visit every year - mandatory. And not just for woodstoves but any heating appliance e. g. your oil burner or gas furnace.


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Location: Going to SFO, the Marin Airporter stops at the Golden Gate Transit bus pad on southbound Highway 101 at the Seminary Drive exit. Returning from the airport the bus drops off at the northbound exit.
Departures: Buses pick up at Seminary Drive every 30 minutes, at approximately :05 and :35 minutes past the hour, from 4:05 AM until 11:05 PM. The trip to SFO takes a minimum of 55 minutes, but it can take longer depending on traffic conditions.
Fares: One-way fare from Seminary Drive is $22 per adult, children 12 and under FREE with a full fare adult (limit 2 children per adult). The fare may be paid directly to the driver of the bus. ALL FARES ARE CASH ONLY ! We cannot accept payment by credit card. No reservations are needed.
Parking: Parking at Seminary Drive is limited to street parking and subject to posted limits.


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We are, in a sense, never in "possession" of the figure depicted in this painting. When viewing a nude figure, especially one whose face is exposed, thereby revealing their identity, the audience may feel they are in a position of power or entitlement. I was once told by a fellow artist that paintings which depict a figure staring back at the viewer do not sell as well as paintings in which the figure does not meet the viewer's gaze. Again, I think this has to do with a voyeuristic power the viewer may enjoy. The raven-headed figure, however, evades us at every turn. On one side, the figure challenges us but remains anonymous. On the other side, we could have glimpsed the face, we might have been satisfied by gazing at the figure's buttocks, but we are spurned. The figure flees, and we are left wondering, "What was that?"
It eventually became clear that this piece is also about gender. As I publicly documented the progress of this piece via social media, I referred to it as the "Raven Queen", and I encountered a handful of people who confessed that they did not know why I was calling the subject of my painting a "queen" because, to their eyes, my subject was male. I felt a mixture of indignation and puzzlement at this reaction because on one side of the piece the subject is clearly depicted with a female breast, albeit small, and because the subject is me. It is true that I am uncommonly muscular, with small breasts, but I was born female and I continue to identify as a woman. Yet because my viewers could not see my face, nor my genitalia, I was perceived as male. I pointed out to a few individuals that the male pectoral structure does not at all look like that of the Raven Queen's, and while she is well-defined, her muscular structure is not that of a man's. But I see these things because I have a trained eye, accustomed to the intricate variations found in the nude figure. You, the viewer, despite your best intentions, are relying on preconceived notions. I am sure that I am guilty of this as well in other contexts. We would all do well to acknowledge the fact that we are all the same, that we are all different, and that we may think we understand what we are seeing, but our gaze is often distracted and superficial. Learning to draw and paint, after all, is just learning to truly see.


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|Reviews for The Mongoose Conspiracy
| pipinheart chapter 3 . 5/11/2016
Loved it.. That is something you never would have thought they would see.
| Voldemort- Coolest Badguy EVER chapter 3 . 5/28/2010
Here we are at the end of another story. It's been so long since I've read and reviewed, but this was a freaking awesome story to start off with.
Seriously, this has been one of the funniest stories ever, not only with its random (and strangely REAL) story matter, but also the usual humour that you and Ridley can slip into your stories.
Like Dean's line: "You let Bobby baby-sit us?" You gotta love that THIS was all he took out of the story. 8P
And, of course, Jeff's concluding lines for the story: For a mongoose, he is pretty cool.
Thanks for a random but incredible story!
- Coolest Badguy EVER
| Voldemort- Coolest Badguy EVER chapter 2 . 5/28/2010
It has to be said again - this is the weirdest story ever. And how I love it. 8)
I got quite a giggle here:
Dean raised his eyebrows. "A mongoose is sitting in your La-Z-Boy watching NASCAR and this was normal to you?"
"He didn't watch NASCAR!" Bobby exclaimed. He lowered his voice and clarified his statement. "He watched movies."
And I got such a nice laugh at the image of Pastor Jim opening the confessional door, being introduced to a talking mongoose, and then calmly shutting the door again. Oh Jim, you are so awesome. 8)
And I loved the Mafia references at the end as well as Jim's long-suffering comment: "The Brotherhood is not linked to the Mafia, as I have told you many times."
Awesome chapter.
- Coolest Badguy EVER
| Voldemort- Coolest Badguy EVER chapter 1 . 5/28/2010
This has to be one of the funniest, most random stories you've written!
Naturally, when I read that the story of Geoff was a true and documented one, I figured you were messing with me. But, lo and behold, Wikipedia introduced me to the full legend of "Gef: The Talking Mongoose".
You gotta love the world we live in. 8)
- Coolest Badguy EVER
| funkyhigh chapter 3 . 1/30/2008
lol, strange
| Savage Shade chapter 3 . 10/22/2007
Hahaha, I couldn't stop laughing in some of this. Great work.
| Savage Shade chapter 1 . 10/15/2007
Hahaha, glad to see you keep your word, after all you did say it'd be funny. Good start.
| gretchless chapter 3 . 10/11/2007
yup, i think that is defo the weirdest brotherhood story i have ever read thats for sure !
it was funny like ... i liked the 2i should have never answered the phone' from john and the "Neurology. I work with brains. Why is it so hard to remember that I'm not a veterinarian?" hehe !
cant wait to see the dog that won ! but i do have a question ? if a new dog is coming into it wont to be on jim's farm ? who looks after jims farm when caleb, dean and sam are all hunting and mac is working ?
bye for now !
| cavetrollgrl chapter 3 . 10/9/2007
Bobby's right quoting Old Yeller is low. The boys will never believe the triad and bobby now. Jeff getting the last word in was teriffic. a Mongoose flipping channels like a bored teenager. Please keep jewels like this coming.
| WindStar chapter 3 . 10/9/2007
this made me laugh so much thanks!
| sams1ra chapter 3 . 10/9/2007
Hee. *sniggers*
And hey, again with the no alerts. It's weird, you're the only one I'm not getting alerts from... *ponders*
| Winchester73 chapter 3 . 10/8/2007
I really enjoyed this story! I chuckled over the way the Triad was conned - Thanks for the ride!
| Angelustatt chapter 3 . 10/8/2007
That was sheer brilliance! I haven't laughed so hard in ages, guys and I'm talking the tears in the eyes kind of laughing. Seeing the Triad beaten by a mongoose is the stuff of legend. LOL
Well done!
| TraSan chapter 3 . 10/8/2007
Ahahahahahahaha...I finally caught up tonight. :D
What a fun story. Sometimes it's just really good to laugh (even if it is at the Triad's expense).
Thanks to you both for writing!
| Becomingwhaturmeantobe chapter 3 . 10/8/2007
That was great. Loved it! It's nice to see a funny story once in a while!


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- Main article: Crown Store
- "Expands your inventory by 1 slot per lesson. Mount carry capacity allows you to carry more items."
- ―In-game description
Crown Lesson: Carry Capacity is an upgrade available for purchase from the Crown Store. This pack includes 10 consumable items that increases mount carrying capacity without the daily limit found at stables. This upgrade requires at least one mount, and can not be used to exceed level 60.


text:
I am caring, friendly and approachable with a conscientious, hardworking and can do attitude towards work. l have excellent interpersonal, time keeping and organisational skills and can effectively communicate with people on all levels. I have a flexible approach and can deal with situations with tact and sensitivity. I have experience working as carer and in an administration role supporting others and team leadership. Any task I undertake I always complete to the very best of my ability. I am happy to participate in any additional training and development required. I am available for immediate work.
Live In Care Experience: 6+ Yrs. Yrs
General Care Experience: Live in care work, Home Care, Care for close relative or loved one
Gender: Female
Looking for work in the following Locations:
Other Information
English Language Level: Advanced
Driving Licence: No Licence
Care Qualifications:
Care Skills: Complex Care, Dementia, Learning Disability, Manual Handling, Personal Care, Stroke
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text: i am currently using a G1 and i want to upgrade. in short i really see that i have two options.. 1. Buy a mytouch slide off the streets or internet for probably around $250-300 2. Make the big switch over to Verizon and pick up a droid 2 for about $200 + sign up fee's i want a new style android phone with a full keyboard and i dont have any upgrades on my t-mobile plan (I'm also not in-contract) what would you do???


text:
Written as a letter to her children, Kelly Corrigan's Lift is a tender, intimate, and robust portrait of risk and love; a touchstone for anyone who wants to live more fully. In Lift, Corrigan weaves together three true and unforgettable stories of adults willing to experience emotional hazards in exchange for the gratifications of raising children.
Lift takes its name from hang gliding, a pursuit that requires flying directly into rough air, because turbulence saves a glider from sinking out. For Corrigan, this wisdom becomes a metaphor for all of life's most meaningful endeavors, particularly the great flight that is parenting.
Corrigan serves it up straight: how mundanely and fiercely her children have been loved, how close most lives occasionally come to disaster, and how often we fall short as mothers and fathers. Lift is for everyone who has been caught off guard by the pace and vulnerability of raising children, to remind us that our work is important and our time limited.
Like Anne Morrow Lindbergh's Gift from the Sea, Lift is a meditation on the complexities of a woman's life, and like Corrigan's memoir The Middle Place, Lift is boisterous and generous, a book readers and listeners cant wait to share.
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Listened to it twice! Great quick listen!
This is a book I always have on my iphone. Great to share with others on a long car ride or 2 hour flight...I love the stories she tells. Not sure it would mean as much if I had not read her first book prior, which I highly recommend..another great story.
This story is so real...she can tell it like it is! It makes you laugh, and cry!
Yes...love all of them!
If you are a mom...take the time to read...and reflect on your own kids!
- sande
Weird mom venting
- Jennifer T. Haley


text: Hi, Bulldog. Thanks for starting a new post, as I suggested. Did you see cj7's response to you in the other thread...the one titled Autoimmune Hepatitis? It's on the 2nd page. She explained how she got pregnant (not intentionally) and had to abort at 3 mos. Please do not continue trying to get pregnant until you have discussed this thoroughly with your hepatologist.