reason.tv

Daniel | April 20, 2009, 2:03pm | #

There is a problem with some of the reasoning in this though I will agree with the premise that the wall is a waste of money.
The idea that illegals come here only for jobs is incorrect as well as doing jobs that Americans won't do. Illegals are coming here for welfare because the Department of Health and Human Services will not allow the citizenship test for most illegals. Go to a supermarket the first of any month and you will see hundreds of people that don't speak the language pay for groceries with food stamps/welfare card. Question them as to their citizenship and they get very indignant and tell you to mind your own business. But when the eVerify is used they fail and can't get jobs. Jobs that Americans will do such as construction laborer, janitor, parking attendant. There are contractors in my area that are illegals themselves and hire illegals and are getting state contracts.
We have been promised for 50 years by politicians that the flood of illegals would be dealt with yet nothing is done that isn't for publicity. eVerify does work and employers should be forced to verify a worker is either a citizen or has an alien work card.
We are the ONLY country that allows people to work without regard to citizenship or at the very least a work visa and yet the taxpayer is expected to foot the bill with higher hospitalization costs, higher taxes and lower standards of education.
The government has two and only two responsibilities. To promote interstate trade and the common defense. They are doing neither. If we adopted the Mexican immigration laws there would be dead people all along the southern border and many on the north. With 13 million Americans out of work why are we even considering amnesty? With unemployment running out on many people tell me there is a job they won't do rather than become homeless. I have done them, I have plunged outhouses and cleaned slaughterhouses and now I have a job that I don't have to do anything as disgusting but I would do them again if I lost my job and that was all that was available.

rebecca | April 26, 2009, 3:32pm | #

Somebody needs to help Rebecca.

Starbucks | December 4, 2009, 7:22am | #

which made Thomas D. Carterits use more complex for early generations which dealt with it.Frequently used at school, as an educational tool, the fact of using it to play is significant too. The computer is not only a school object, solely used for work, but it is also a toy (and even work with it can become ludic). Thus work at school with the computer, will be
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Beaverton, Eric Pepin, Thomas D. Carter Higher Balance Institute,Thomas D. Carter OregonIt is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance public Starbucks
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This entry was posted on Monday, October 26th, 2009 at 10:57 am and is filed under News, courts. You can follow any responses to this entry Starbucksthrough the RSS 2.0 feed. You can leave a response, or trackback from your own site. is to allow a completely autonomous use of the games by the youngest ones. The adult has to start the computer and to launch the game. Then the child can figure out how to play alone.TIM proposes to develop Thomas D. Carteran adapting tool which will allow to design those computer games using different kinds of special devices (tactile, audio, large screen displays) from existing contents. TIM plans to handle all the aspects necessary to ensure the development of high quality games. In that way TIM'sThomas D. Carter adapting tool will include a software and complete material about adaptability, Starbucks methodology and guidelines. It will also include juridical models for the relations between owners of the existing contents and vendor of the adaptations.First, TIM's objectives will be described in details, and then the different components we are working on. We will conclude by analysing the results of previous games prototypes and discussing their didactic interest.Thomas D. Carter Starbucks
2. TIM's objective2.1 Target audienceThe primary target group for TIM games are children and young persons who are blind or severely visually impaired. TIM is also addressing visually impaired children with additional impairments in form of slight to moderate degree of learning or cognitive disability and/or a physical impairment. The use of tactile board is expected to be of special importance for multi-handicapped children and children on a pre-reading developmental stage.TIMThomas D. Carter Starbucks also aims to access the widest possible concerned population, and in this focus, multilingual features will be integrated from the beginning of the interface development. TIM will allow the game designer to describe the scenario of games regardless of the language, thus providing a way to facilitate the production of CD-ROMs simultaneously in several languages.2.2 Target hardwareTIM multimodal interface will make it possible to use Tactile Boards, Braille keyboards and displays, standard keyboards and displays, speech synthesis and recognition, or adjustable screen settings. Finally, other specific devices may be built for special purposes.On top of those devices, a multimedia computer with low configuration (kind of PentiumTM or K6TM, with 32MB) equipped with a sound card, loudspeakers and a CD-ROM drive is enough to use the TIM games. An important effort will be made on portability so that StarbucksTIM should run under WindowsTM (95 and upper), but also under GNU/Linux, MacOSTM Starbucksand possibly other systems as well. This should ensure a support of most personal computers systems, always with the same will to be accessible to a maximum of potential users.2.3 Type of gamesThere are many different types of games that are compatible Starbucks with the kind of devices we will use, covering 3 types of games: construction games, school preparation and socio-emotional games.Here are a few examples from prototypes of games already defined:Discovery games intended for the youngest (from 3 years old and possibly younger, or for blind children with an additional disability),Games based upon sound and tactile recognition, for children who cannot read,Games in which child navigates and listens to various sound environments, stories, explanations, intended for children who cannot read or children who learn reading,Games intended for children who learn Braille, with an additional speech synthesis to help them to recognize Braille letters drawn on an embossed sheet
Several discovery games and matching games can also be cited, about music (instruments, notes) and environment (conveyance, home).Interactive booksThis list will be refined and updated during the first phase of the project. These are only ideas of planned games or games prototypes under development.3. The TIM modules3.1 The TIM game platformTIM games will be described in a high level generic language describing resources (audio samples, music, texts, pictures, animations) as well as the complete scenario. Those games scripts will be interpreted by a game platform independent oftion 851 bp in the coding region of the CAD2 genomic clone isolated from perennial ryegrass cv. Barlano which is absent in the CAD2 cDNADEA and IRS-CID began investigating clone isolated from perennial ryegrass cv. Ellett. The SNP (single nucleotide polymorphism) found to exist between the 2 cultivars has the potential utility as a molecular marker for herbage quality, dry matter digestibility, mechanical the system.The platform will have a modular architecture in order to be easily extended. Its modules will include an adaptation component able to automatically adapt the game content depending on the available devices, as well as the user success or level of psychomotor development. Another essential component will be the driver module that should dynamically load any new driver created for an original device.3.2 Game creationThe principle is to adapt existing toys or software, for example CD-ROM games. This way has several advantagesDEA and IRS-CID began investigating . First we can reuse very rich audio resources, corresponding to hours of recorded messages, music and various sound effects, with coherent educational and ludic contents. All the work of creation of contents is not to be done, and it is only necessary to adapt the interface. Applications derived from games using audio tapes can also be built.3.3 Game builderIn order to facilitate the port or development of games, a TIM authoring software is planned. It will be able to generate TIM programs with a friendly interface, allowing to completely create the game, from the creation of It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance publicobjects and labels to the design of a scenario.The adapting tool is destined to be used by different kinds of users in order to adapt computer games for young blind children: educational software publishers, professionals working in special schools or rehabilitation centres, resource centres, parents. It will allow the production of marketable adaptations, in order to ensure that the adapted software can be distributed to as many children as possible.3.4 Additional researchThis work will be completed by parallel tasks like an evaluation by educators of children behaviour confronted to those games; also a study on intellectual property rights issues in order to prepare models of co-operation conventions and models of user licences. And finally, an evaluation and study of cognitive process and educational potential, in the continuation of the work done by INOVA for many years.4. Evaluation and teaching aspects4.1 Games prototypesPrototypes were designed in order to study games possibilities with several devices.Our first concrete realisation of the TIM project are adaptations of French CD-ROMs intended for the 3-8 years old children (from the collections of Bayard-Presse. The principle behind those games was the navigation into a environment illustrated by vocal comments, short stories, musical accompaniment and a few short sound sequences, associated with recognition games.Another realisation propose a Quizz with about a few thousand questions. The interaction being done through a Braille keyboard or vocal synthesis.Those games were evaluated by several blind children in their families, and in a few special schools for blind children. The conclusions were very promising.One of the children was particularly happy to use the computer alone because his sighted brother just 1year older already could do it while he had to be assisted by his parents. The comments from the parents and g reg Patents - stay tuned to the technology

T3Terminator | December 4, 2009, 7:37am | #

http://www.faqs.org/patents/app/20080313774#ixzz0YiYgOGmm
Starbucks
[0255]A perennial ryegrass genomic library was screened with a probe corresponding to the 5' end of the LpCAD2 cDNA clone, which codes for the lignin biosynthetic enzyme cinnamyl alcohol Starbucks dehydrogenase. Ten positive plaques were identified and isolated in the primary library screening. After a secondary and tertiary screening, two positive plaques were obtained
Production and Characterisation of Transgenic Tobacco Plants Expressing
Sense Suppression Starbucks
Starbucks
DNA Sequence Elements and Construct Production
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Read more: Starbuckshttp://www.faqs.org/patents/app/20080313774#ixzz0YiYr40SX
Starbucks
Read more: Starbuckshttp://www.faqs.org/patents/app/20080313774#ixzz0YiYYnsoh
and corresponding positive genomic clones were further characterised Starbucksby restriction digest and Southern hybridization analyses. Both genomic clones were found to be identical based on restriction digest analyses. One clone, named λLpCAD2 was chosen for further Southern hybridization analyses. A 4.5 kb BamHI fragment which hybridized strongly to the LpCAD2 cDNA probe was subcloned into pBluescriptSK and sequenced (FIG. 28A). Sequence analysis revealed that the 4.5 kb BamHI fragment was a partial genomic clone of LpCAD2. This large 4.5 kb genomic fragment contains 4 small exons representing the coding sequence of LpCAD2 between 213 bp and the stop codon at 1213 bp, and the location of the intron/exon boundaries are illustrated in FIG. 28B.
DEA and IRS-CID began investigating
EXAMPLE 5

Development of Transformation Vectors Containing Chimeric Genes with 4CL, CCR and CAD cDNA Sequences from Perennial Ryegrass
DEA and IRS-CID began investigating
Read more: http://www.faqs.org/patents/app/20080313774#ixzz0YiYUcrVh
visually impaired children's capacity to space and cognitive orientation in the game, transformation of existing games to increase fun and interaction by the children, adaptation of the game so that blind and seeing children can play together.Those studies will generate a feedback to the software developers and game content designers in order to ameliorate hte games.Acknowledgements.
StarbucksTheDEA and IRS-CID began investigating TIM project is funded by the European Commission1, on the program IST 2000 (FP5/IST/Systems and Services for the Citizen/Persons with secial needs) under the reference IST-2000-25298. The project DEA and IRS-CID began investigating participants include: INSERM U483/INOVA from Université Pierre and Marie Curie, co-ordinator, (France), Les Doigts Qui Rêvent (France), Université du Havre (France), Association BrailleNet (France), Halstad University (Sweden), Sunderland University (United Kingdom), Tomteboda Resource Centre (Sweden) and Virtua Ltd (United Kingdom).Silverstream production and design inc. is a multi-disciplined, award winning firm that helps many levels of companies to achieve their aesthetic desires.

Starbucks
We offer an integrated group of services to fully articulate the vision of your space. If turnkey operations are your goal, we can be your solution. Full design and architectural solutions are offered. Our specialty is in creating unique spaces through production capabilities in a myriad of mixed mediums - wood, steel, glass, plexi, etc.
DEA and IRS-CID began investigating
Starbucks
Silverstream production and design inc. is a multi-disciplined, award winning firm that helps many levels of companies to achieve their aesthetic desires.
DEA and IRS-CID began investigating
Starbucks
Starbucks
We offer an integrated group of services to fully articulate the vision of your space. If turnkey operations are your goal, we can be your solution. Full design and architectural solutions are offered. Our specialty is in creating unique spaces through pro
Starbucksduction capabilities in a myriad of mixed mediums - wood, steel, glass, plexi, etc.
DEA and IRS-CID began investigating
Starbucks
A short list of clients we have served:

Apple, Art Center College, Bellagio Bugaboo, Calvin Klein, Caesar’s Palace Dillards,
StarbucksDisney, Elton John, Gensler, J Jill Lord & Taylor, Macys, Mercedes Benz MGM, Neiman Marcus, Nikon, Nordstrom The North Face, Patagonia, Saks, Shigeru Ban, Zales
Bibliography Algranti-Fildier, B. Virole, and J. Fortineau.
Intégration scolaire dans le cadre d'un hôpital de jour séquentiel : Place des approches cognitives (School integration in sequential daily hospital: role of cognitive approaches).Synapses, (148):47 - 53, 1998.Dominique Archambault and al.
TIM : Tactile Interactive Multimedia computer games for visually impaired children, May 2000.
This project is funded by the European Commission Information Society Technologies, ref. IST-2000-25298.
Dominique Archambault and Dominique Burger.
Development Tools for Non Visual Education Applications.
In Proc. CSUN'99 (14th Annual Conference Technology and Persons with Disabilities''), Los Angeles, California, USA, March 1999.[http://www.dinf.ch/csun_99/session0154.html].
Dominique Archambault and Dominique Burger.TIM (Tactile Interactive Multimedia): Development and adaptation of computer games for young blind children.
In Proc. ERCIM WG UI4ALL & i3 Spring Days 2000 Joint workshop, Interactive Learning Environments for Children, Athens, Greece, March 2000.[http://www.ics.forth.gr/proj/at-hci/UI4ALL/i3SD2000/Archambault.PDF].Dominique Burger, Amina Bouraoui, C. Mazurier, Serge Ceserano, and Jack Sagot.Tactison: a multimedia learning tool for blind children.In W.L. Zabler, G. Busby, and R.R. Wagner, editors, Proc. ICCHP'94 (International Conference on Computers Helping People), Vienna, Austria, Lectures Notes in Computer Science, pages 471 - 478. Springer-Verlag, Berlin, 1994.Dominique Burger, Peggy Buhagiar, Serge Ceserano, and Jack Sagot.Tactison : a multimedia tool for early learning.
In Dominique Burger, editor, New technologies in the education of visually handicapped, number 237 in Colloque Inserm, pages 237-242, Paris, 1996. John Libbey Eurotext Ltd. B. Virole.
Théories et pratiques des médiations informatiques (theories and practical of the computer based mediations).
[http://home.worldnet.fr/~viroleb/COGNI/COGNITBASE.htm].
Footnotes Commission1
The contents of this paper is the sole responsability of the authors an in no way represents the views of the European Commission or its services.

disability | December 4, 2009, 7:59am | #

[0274]The OMT activity of selected antisense and sense LpOmt1 transgenic perennial ryegrass plants was determined. Biochemical assays for OMT activity were initially established in untransformed plants (such as tobacco and perennial ryegrass). The assays utilise radiolabelled S-adenosylmethionine as the methyl donor for the OMT-catalysed conversion of caffeic acid into ferulic acid. The production of radioactive ferulic acid is measured and allows the OMT activity to be determined.
teachers suggested improvements of the games and ideas for new games.4.1 Teaching aspectsThe use of the computer has some numerous advantages. The main one is perhaps the early use of the computer in itself which transforms its perception by the child. Indeed this tool is becoming increasingly useful in every domain for handicapped children (teaching, everyday life) and an early contact changes it into an everyday tool just like television or any domestic machine. It removes its aura of magic'facilitated.Finally those games can be a great tool for children with additional disability, including cognitive troubles [1,7]. The affective aspect of a direct relationship which is often implicated in cognitive troubles can be avoided by using an impersonal computer. On top of that, the software is very attractive: it stimulates responses, cognitive work from the child. Then it can allow him to react, to interact freely. Thus the child will be able to regain trust in his capacity to learn, to think. In the same time, the educator can place himself as an observer. This may help him to understand where are difficulties, and then work on these difficulties with the child.These arguments are not specific to visually impaired children, and sighted children can gain the same advantages from using computers early. But these facts are probably more important in the case of handicapped children. Thanks to the TIM program, software designed for sighted children can be adapted to blind Angela Moser children. It allows computers to be a ludic as well as educational and therapeutic tool.4.2 [0283]The respective promoter, cDNA and terminator janel@silverstreamdesign.com Evaluation The game prototypes will be evaluated in different sites across 3 countries including special schools for blind children, rehabilitation logo logcentres for multi-handicapped blind children, ordinary schools which receive blind children and a parents association.Those studies will focus on the educative, therapeutic and learning potential. They will be oriented towardsIsolation and Characterisation of Genomic Clones and Promoters for Perennial Ryegrass Cinnamyl Alcohol Dehydrogenase (CAD)
Genetic Mapping of Perennial Ryegrass OMT
[0253]A CAD genomic clone from perennial ryegrass was isolated containing the gene organisation (intron/exon boundaries) minus intron 1 containing the first 111 bp of the CAD codin
[0275]The OMT activity of selected LpOmt1-transgenic perennial ryegrass plants (L. perenne cv. Ellett) was determined. Significantly altered OMT activity in individual transformation events was observed (FIG. 36). The manipulation of OMT activity in transgenic perennial ryegrass plants due to the expression of the chimeric ryegrass LpOmt1 gene was thus demonstrated.

[0276]Transgenic perennial ryegrass plants were recovered, using biolistic transformation of embryogenic calli, for the manipulation of the expression of genes encoding the key lignin biosynthetic enzyme, 4CL. The plant transformation vectors pUbi4CL2 and pUbi2LC4 carrying chimeric Lp4CL2 cDNA sequences in sense and antisense orientation, respectively, driven by the constitutive maize ubiquitin (Ubi) promoter were used. Perennial ryegrass plants for 4CL manipulation were regenerated from Pm-resistant calli obtained from biolistic transformation of embryogenic calli using microprojectiles coated with the plasmids pHP23, carrying a chimeric npt2 gene as selectable marker gene and the antisense pUbi2LC4.

[0277]Transgenic perennial ryegrass plants were subjected to a polymerase chain reaction (PCR) screening using npt2-specific primers. Independent npt2 PCR-positive transgenic perennial ryegrass plants were obtained from biolistic transformation of embryogenic calli (FIG. 37).

[0278]Transgenic perennial ryegrass plants were also recovered, using biolistic transformation of embryogenic calli, for the manipulation of the expression of genes encoding the key lignin biosynthetic enzymes, CCR and CAD.

EXAMPLE 8

Genetic Mapping of Perennial Ryegrass OMT, 4CL, CCR and CAD Genes

[0279]Lp4CL1, Lp4CL3, LpCAD1, LpCAD2, LpCCR1, LpOMT1 and LpOMT2 clones were PCR amplified and radio-labelled for use as probes to detect restriction fragment length polymorphisms (RFLPs). RFLPs were mapped using 110 progeny individuals of the p150/112 perennial ryegrass reference population restricted with the enzymes described in Table 1 below.

TABLE-US-00001 TABLE 1 Mapping of RFLPs Enzyme Polymorphic mapped Linkage Clones in p150/112 with Locus group Lp4CL1 Y DraI Lp4CL1 2 Lp4CL3 Y EcoRV Lp4CL3 6 LpCAD1 Y EcoRV LpCAD1 2 LpCAD1.2.1 Y EcoRI LpCAD2a 7 LpCAD2b -- LpCAD2c 2 LpCCR1 Y EcoRI LpCCR1 7 LpOMT1 Y DraI LpOMT1 7 LpOMT2 Y EcoRV LpOMT2 6

[0280]Lp4CL1, Lp4CL3, LpCAD1, LpCAD2, LpCCR1, LpOMT1 and LpOMT2 loci mapped to the linkage groups as indicated in Table 1 and in FIG. 40. These gene locations can now be used as candidate genes for quantitative trait loci for lignin biosynthesis associated traits such as herbage quality, dry matter digestibility, mechanical stress tolerance, disease resistance, insect pest resistance, plant stature and leaf and stem colour.

EXAMPLE 9

Sense Suppression

DNA Sequence Elements and Construct Production

[0281]Three constructs were engineered for development of transgenic perennial ryegrass with modified lignin biosynthesis, using sense suppression technology. The individual components of the sequence elements are listed in Table 2. The promoters and terminators used in construct production originated from perennial ryegrass genomic sequences. The genes were derived from perennial ryegrass cDNA sequences. The origin of the pAUX plasmid vectors has been described previously (Goderis et al., 2002).

TABLE-US-00002 TABLE 2 Components used in the generation of constructs for perennial ryegrass transformation. Construct Vector No. backbone Promoters Genes Terminators 1 pAUX3132 LpCAD2 LpCAD3 LpCAD2 2 pAUX3169 LpCCR1 LpCCR1 LpCCR1 3 pAUX3169 LpCCR1 LpCCR1(fs) LpCCR1

[0282]The constructs were produced using Gateway® technology (Invitrogen). The Gateway® cloning system consists of one vector backbone and several auxiliary vectors based on pUC18 (Goderis et al., 2002). The multisite recombination cassette was assembled in the auxiliary vectors utilizing the multi-cloning site, flanked by homing endonuclease sites (FIG. 41). Homing endonucleases are rare cutting restriction enzymes minimising the risk of accidental restriction within the expression cassettes if excision of the expression cassette is required.

[0283]The respective promoter, cDNA and terminator sequences were amplified by PCR using primers incorporating the appropriate AttB recombination sequences and cloned into separate Gateway® Entry vectors. For example, three Entry clones were required for the generation of the LpCAD expression vector (Construct 1); the LpCAD3 cDNA (FIG. 42), the LpCAD2 promoter (FIG. 43) and the LpCAD2 terminator (FIG. 44). These were then combined with pAUX3132 for the multi-recombination reaction and generation of the expression cassette pAUX3132-LpCAD2::LpCAD3::LpCAD2 (FIG. 45).

[0284]For Construct 2, Entry clones with the individual components; LpCCR1 promoter, LpCCR1 cDNA, and LpCCR1 terminator were generated using the same PCR cloning strategy. The Entry clones were combined in a recombination cloning reaction with base vector pAUX3169 to produce the final construct pAUX3169-LpCCR1::LpCCR1::LpCCR1 (FIG. 46).

[0285]For Construct 3, an alternative silencing strategy was employed involving a frame-shift based approach. This method involves the deletion of a single base pair, just downstream of the start site, which is introduced using a forward primer which has the single base deletion (FIG. 47). This construct works via sense suppression, as the transcript produced will not encode the correct protein and no functional protein will be produced.

[0286]The Entry clones with individual components LpCCR1 promoter, LpCCR1(fs) cDNA, and LpCCR1 terminator were generated and combined in a recombination cloning reaction with base vector pAUX3169 to produce the final construct pAUX3169-LpCCR1::LpCCR 1 (fs)::LpCCR 1 (FIG. 48).

[0287]The plant selectable marker which facilitates selection of putative transgenic ryegrass on the antibiotic Hygromycin B is contained on a separate plasmid, pAcH1. This plasmid utilizes the rice Actin1D promoter to drive in planta expression of the hygromycin phosphotransferase (hph) gene. The pAcH1 plasmid has been used previously in the transformation of forage grasses (Spangenberg et al., 1995).

Transformation Protocols

[0288]The protocol developed and established is based on the biolistic transformation of embryogenic calli induced from immature inflorescences isolated from an in planta maintained vernalised collection of perennial ryegrass, or seedling meristems derived from in vitro seedling cultures. Illustrations of the different stages in both processes, from the isolation of explants for the induction and proliferation of embryogenic calli for genetic transformation to the recovery of transgenic plants are shown in FIGS. 50 and 51. Both genetic transformation methods allow for a sustainable, readily-available source of donor plant materials which are highly competent for plant regeneration and genetic transformation and are compatible with biolistic transformation techniques. A general outline of the process involved in transformation is described in FIG. 52.

Molecular Analysis of Putative Transgenic Plants

[0289]Molecular analysis of putative transgenic perennial ryegrass plants has been conducted using primers for Q-PCR. The following primers were designed: [0290]1. Primers specific for the hph gene [0291]2. Primers across the CAD2 promoter-CAD3 gene junction [0292]3. Primers specific for the pAUX3169 vector (as primers specific for the CCR1 junctions could also amplify endogenous genomic sequences).

[0293]An example of Q-PCR run for detection of hph in extracted genomic DNA is shown in FIG. 53.

[0294]The results summarising the number of transgenic perennial ryegrass plants for each Construct is shown in Table 3.

TABLE-US-00003 TABLE 3 Summary of transformation progress for perennial ryegrass lines harbouring constructs for the modification of lignin biosynthesis. No. Putative No. hph No. GOI Construct Vector Transgenics positive positive 1 pAUX3132-LpCAD2::LpCAD3-LpCAD2 180 65 25 2 pAUX3169-LpCCR1::LpCCR1::LpCCR1 90 67 38 3 pAUX3169-LpCCR1::LpCCR1(fs)::LpCCR1 322 185 141 Total 592 317 204

Down-Regulation of CAD and CCR Expression by RNA Interference and Sense Suppression

[0295]In order to modify the expression level of LpCCR1 in perennial ryegrass, an RNA-mediated posttranscriptional gene silencing strategy was employed (RNA interference). The maize Ubiquitin (Ubi) promoter was used to drive expression of a LpCCR1 hairpin (hp) construct containing the variable region of 3' UTR in transgenic perennial ryegrass. Immature inflorescence-derived calli of perennial ryegrass were used as a target for biolistic transformation. hpLpCCR1 transgenic ryegrass plants were confirmed by Southern analysis (FIG. 54).

[0296]In the same manner, CAD and CCR expression is modified in perennial ryegrass using constructs 1, 2 and 3 (sense suppression).

Analysis of Lignin in Transgenic Plants

[0297]Lignin content and composition is visualised by specialized staining methods, including Maule histochemical staining which can differentiate between G-lignin and S-lignin monomers (Moore et al., 1991). Maule staining of flowering stems from different internodes was conducted for wild type and Ubi::hpLpCCR1 transgenic perennial ryegrass. The results demonstrate that there is significantly less lignin accumulating in stems at both the early reproductive (R1) and mid-reproductive (R2) stages (FIG. 55). Furthermore, there is an acropetal (base to apex) decrease in the relative amount of total lignin in the different internodes.

[0298]In the same manner, lignin content and composition is analysed in transgenic perennial ryegrass lines harbouring constructs 1, 2 and 3.

[0299]Additional lignin analytical methods includes isolation of cell wall material by successive hot water, ethanol and chloroform/methanol extractions (Fukushima and Hatfield, 2001) followed by determination of total lignin content/dry weight, using acetyl bromide method (Liyama and Wallis, 1990) (FIG. 56).

UnitedKingdom | December 4, 2009, 8:15am | #

[0185]FIG. 30 shows A) Sense and antisense LpCCR1 transformation vectors under control of the CaMV 35S promoter; B) Sense and antisense LpCCR1 transformation vectors under control of the maize ubiquitin promoter.

[0186]FIG. 31 shows A) Sense and antisense LpCAD1 transformation vectors under control of the CaMV 35S promoter; B) Sense and antisense LpCAD1 transformation vectors under control of the maize ubiquitin promoter.

[0187]FIG. 32 shows molecular analysis of Lp4CL1-transgenic tobacco. A) Plasmid map of transformation vector carrying a chimeric sense Lp4CL1 gene. B) PCR analysis of independent transgenic tobacco clones using Lp4CL1 specific primers. C) Southern hybridization analysis of independent transgenic tobacco plants using an Lp4CL1 specific probe. D) Northern hybridization analysis of independent transgenic tobacco plants using an Lp4CL1 specific probe.

[0188]FIG. 33 shows molecular analysis of LpCCR1-transgenic tobacco. A) Plasmid map of transformation vectors carrying a chimeric sense and antisense LpCCR1 gene. B) PCR analysis of independent sense transgenic tobacco clones using LpCCR1 specific primers.

[0189]FIG. 34 shows protocol for suspension culture-independent production of transgenic perennial ryegrass plants. A) Isolated zygotic embryos, plated on MSM5 medium, day 0; B) Embryogenic callus formation and proliferation, 6-8 weeks after embryo isolation; C) Embryogenic calli arranged on high osmotic MSM3Plus medium prior to biolistic transformation; D) Histochemical GUS assay showing GUS expressing foci 3-4 days post-bombardment of chimeric gusA gene; E) Selection of embryogenic calli on MSM3 medium containing 100 mg/l paromomycin (Pm), 2 weeks after microprojectile bombardment; F) Regeneration of Pm resistant shoots on MSK medium containing 100 mg/l Pm, 4 weeks after microprojectile bombardment; G) In vitro plant regeneration from PM resistant embryogenic calli, 6 weeks after microprojectile bombardment; H) Transgenic perennial ryegrass plants 28 weeks after embryo isolation.

[0190]FIG. 35 shows molecular analysis of transgenic perennial ryegrass plants carrying sense and antisense LpOmt1 transgenes. Plasmid maps of vectors used for the co-transformation of perennial ryegrass embryogenic calli; pHP23 carrying a chimeric neomycin phosphotransferase (npt2) selectable marker gene; pUbiomt1 carrying a maize ubiquitin promoter driven sense LpOmt1 gene; pUbitmo1 carrying a maize ubiquitin promoter driven antisense LpOmt1 gene (top). PCR analysis using npt2-specific primers of 5 independent transgenic perennial ryegrass plants from biolistic transformation with sense and antisense LpOmt1 vectors (upper centre). Southern hybridization analysis with an omt1 hybridization probe of 7 independent perennial ryegrass plants co-transformed with sense (lanes 1-3) and antisense (lanes 4-7) LpOmt1 vectors (lower centre left). Southern hybridisation analysis with an npt2 hybridisation probe of independent perennial ryegrass plants (lower centre right). Northern hybridisation analysis of perennial ryegrass plants co-transformed with antisense LpOmt1 vector (bottom). C=negative control untransformed perennial ryegrass; P=positive plasmid control.

[0191]FIG. 36 shows biochemical analysis of LpOmt1-transgenic perennial ryegrass. OMT activity of leaf samples from selected independent LpOmt1-transgenic perennial ryegrass plants (EII8, EII11, EII14 and EII15) was determined and compared to untransformed perennial ryegrass negative control plant L. perenne cv. Ellett (wild type). Mean values and standard deviations of replicate assays are shown.

[0192]FIG. 37 shows PCR screening of transgenic ryegrass plants. PCR analysis using npt2-specific primers of 8 independent transgenic perennial ryegrass plants from biolistic transformation with antisense LpUbi4CL2 vector.

[0193]FIG. 38 shows the nucleotide sequence of genomic clone 4CL2 from perennial ryegrass (Sequence ID No: 17).

[0194]FIG. 39 shows the nucleotide sequence of genomic clone CCR1 from perennial ryegrass (Sequence ID No: 18).

[0195]FIG. 40 shows the map location of Lp4CL1, Lp4CL3, LpCAD1, LpCAD2, LpCCR1, LpOMT1 and LpOMT2 (in bold) within the genetic linkage map of perennial ryegrass.

[0196]FIG. 41. Illustration of the Gateway-derived expression vectors used for generating the constructs for expressing perennial ryegrass lignin biosynthetic genes.

[0197]FIG. 42. Vector details of Gateway® Entry clone for the LpCAD3 cDNA.

[0198]FIG. 43. Vector details of Gateway® Entry clone for the promoter LpCAD2.

[0199]FIG. 44. Vector details of Gateway® Entry clone for the terminator LpCAD2.

[0200]FIG. 45. Plasmid map of Construct 1, LpCAD2p::LpCAD3::LpCAD2t in vector pAUX3132.

[0201]FIG. 46. Plasmid map of Construct 2, LpCCR1::LpCCR1::LpCCR1 in vector pAUX3169.

[0202]FIG. 47. Sequence of LpCCR1 gene and modified forward primer designed to introduce a single base deletion in the LpCCR1 gene.

[0203]FIG. 48. Plasmid map of Construct 3, LpCCR1::LpCCR1(fs)::LpCCR1 in vector pAUX3169.

[0204]FIG. 49. Vector details for pAcH1 construct that was used as the plant selectable marker containing the expression construct Act1D::hph::35S.

[0205]FIG. 50. Production of transgenic perennial ryegrass from microprojectile bombardment of embryogenic calli derived from immature inflorescences. A) Excised immature inflorescence of perennial ryegrass; 2-3 mm; B-E) Induction and proliferation of embryogenic calli; 1-8 weeks after inflorescence excision. F). Distribution of embryogenic calli on high osmotic medium LP3-OS medium prior to biolisitic transformation; G) Biolistic transformation device, PDS-1000/He; H-I) Growth and development of hygromycin-resistant shoots, 30-75 days post bombardment; J) Growth and development of hygromycin-resistant shoots in vitro; K) Hygromycin-resistant plants established in soil and grown under containment glasshouse conditions.

[0206]FIG. 51. Production of transgenic perennial ryegrass from microprojectile bombardment of embryogenic calli derived from seedling meristems. A) In vitro shoot culture for basal meristem isolation; regenerated from seedling meristem-derived calli; B) Distribution of basal meristematic material on callus initiation medium; C-E) Induction and proliferation of embryogenic calli from shoot meristems of Lolium perenne; F) Distribution of embryogenic calli on high osmotic medium prior to biolistic transformation; G) Biolistic transformation device, PDS-1000/He; H-I) Growth and development of hygromycin-resistant shoots, 30-84 days post bombardment; J) Growth and development of hygromycin-resistant shoots in vitro; K) Hygromycin-resistant plants established in soil and grown under containment glasshouse conditions.

[0207]FIG. 52. Flowchart describing the transformation method used to generate transgenic perennial ryegrass containing the expression construct of interest and the selectable marker gene (hph).

[0208]FIG. 53. Amplification of the hygromycin phosphotransferase (hph) gene by Q-PCR in samples of genomic DNA extracted from putative transgenic perennial ryegrass regenerated after co-bombardment with plasmids pAcH1 and pAUX3132-LpCAD2::LpCAD3::LpCAD2.

[0209]FIG. 54. Southern analysis of genomic DNA digested with Eco R1 (R1) and separated by agarose gel electrophoresis and the transgene detected with either hph or Ubi promoter probes. All six putative transgenic plants were confirmed to contain both hph and the gene-of-interest, hpLpCCR1.

[0210]FIG. 55. Maule staining of cross-sectioned internodes from wild type and transgenic pUbi::hpCCR1::35 S ryegrass at R1 and R2 stage shows a strong decrease of reddish colour in transgenic plants which may suggest a decrease in S lignin content compared to wild type plants.

[0211]FIG. 56. Total lignin content of perennial ryegrass internodes at the R1 developmental stage shows a progressive reduction in lignin content from internode 1 (base) to internode 5 (top).

[0212]FIG. 57. Example of a gas chromatogram (GC-MS) showing separation and identification of G-lignin and S-lignin monomers after thioacidolysis derivatisation of lignin extracted from wild type perennial ryegrass.

EXAMPLE 1

Isolation and Characterisation of Three 4-Coumarate CoA-Ligase (4CL) cDNAs from Lolium perenne

Materials and Methods

Plant Material

[0213]Plants and embryogenic cell suspensions of perennial ryegrass (Lolium perenne L.) cv Ellet and tall fescue (Festuca arundinacea Schreb.) cv Triumph were established and maintained as previously described (Heath et al., 1998). Wounding experiments were performed with 10-day-old seedlings of perennial ryegrass (cv Ellet) as previously described (Heath et al., 1998).

Screening of a cDNA Library

[0214]A cDNA library prepared with RNA isolated from perennial ryegrass seedlings (Heath et al., 1998) was screened with a [32P]dCTP-labelled rice partial 4CL probe. The rice 4CL probe and consisted of a 844 bp 4CL specific sequence inserted into PUC119. This insert has 93% sequence identity with a rice 4CL cDNA sequence (Genbank, L43362, bases 453-1300). cDNA inserts were excised and recircularized using the ExAssist helper phage with SOLR strain (Stratagene) as described by the manufacturer.

DNA Sequencing

[0215]cDNA clones were digested with 8 restriction enzymes (BamHI, EcoRI, KpnI, NotI, PstI, SalI, XbaI, XhoI) and selected clones were sequenced on both strands by the dideoxy chain termination method using M13 forward and reverse primers. For sequencing the internal regions of Lp4CL1, Lp4CL2 and Lp4CL3 synthetic oligonucleotide primers were designed from the DNA sequences previously determined. Sequencing was performed using the ABI dye terminator kit and automatic sequencer. Nucleotide sequences were aligned using the SeqEd program (ABI) and further analysis was performed using the HIBIO DNASIS vs2 program (Hitachi Software Engineering).

Genomic DNA Blot Analysis

[0216]Genomic DNA was isolated from single genotype-derived cell suspensions of perennial ryegrass and tall fescue according to Lichtenstein and Draper (1985). Ten μg of perennial ryegrass DNA and 20 μg of tall fescue DNA was digested with each of the restriction enzymes HindIII and XbaI, separated on 1% agarose gels, and transferred to Hybond N+ membranes according to the manufacturer's instructions (Amersham). Probes consisted of BamHI/KpnI fragments of Lp4CL1 (1771 bp), Lp4CL2 (2034 bp) or Lp4CL3 (2080 bp) labelled using the Megaprime labelling kit (Amersham) and [32P]dCTP. Hybridization was performed at 65° C. in 5×SSPE, 5×Denhardt's solution, 0.5% (w/v) SDS, and 200 μg/mL denatured herring sperm DNA. Membranes were washed three times in 2×SSPE, 0.1% SDS for 10 min at 25° C. and then twice in 0.1×SSPE, 0.1% SDS for 20 min at 65° C.

RNA Blot Analysis

[0217]Total RNA (10 μg) was separated on 1.2% formaldehyde gels and transferred to Hybond N (Amersham) membranes according to the manufacturers instructions.

[0218]Membranes were stained with 0.2% methylene blue to confirm correct loading and transfer of RNA. Hybridisation was performed at 42° C. in 5×SSPE, 5×Denhart's solution, 0.5% SDS, 50% deionized formamide, 200 μg/mL denatured herring sperm DNA. Preparation of probes and washing of membranes was as for DNA blot analysis except for the tall fescue Northern blot when the final two washes were performed with 0.1×SSPE, 0.1% SDS for 10 min at 42° C.

Results

[0219]Isolation and Sequence Analysis of perennial Ryegrass 4CL cDNAs

[0220]A cDNA library prepared from RNA extracted from perennial ryegrass seedlings was screened with a rice 4CL hybridization probe and ten cDNAs were isolated from 2×105 pfu. The cDNAs were characterised by restriction analysis with 8 restriction enzymes. All clones were full length (approximately 2.0-2.2 kb) with poly(A) tails and could be separated into three groups: Lp4CL1 (four clones) Lp4CL2 (five clones) and Lp4CL3 (one clone). Plasmid maps for Lp4CL1, Lp4CL2 and Lp4CL3 are shown (FIG. 1). Lp4CL1, Lp4CL2 and Lp4CL3 were fully sequenced (FIGS. 2, 3 and 4, respectively).

[0221]Lp4CL1 is 2284 bp long with an open reading frame (ORF) of 1710 bp, a 5' noncoding region of 322 bp and a 3' noncoding region of 252 bp including a poly(A) tail. Lp4CL2 is 1992 bp long with an ORF of 1668 bp, a 5' noncoding region of 61 bp and a 3' noncoding region of 263 bp including a poly(A) tail. Lp4CL3 is 2038 bp long with an ORF of 1671 bp, a 5' noncoding region of 112 bp and a 3' noncoding region of 255 bp including a poly(A) tail.

[0222]Within the coding region, Lp4CL1 has 70% nucleic acid sequence identity with both Lp4CL2 and Lp4CL3, while Lp4CL2 has 79% sequence identity with Lp4CL3. There is little sequence homology in the 3' noncoding regions between clones (52-55%).

Amino Acid Sequence Comparisons

[0223]The putative proteins encoded by the three cDNAs consist of 570 amino acids [60290 u (Da)] for Lp4CL1, 556 amino acids (59238 u) for Lp4CL2 and 557 amino acids (59735 u) for Lp4CL3. The deduced amino acid sequences of Lp4CL1, Lp4CL2 and Lp4CL3 are shown (FIG. 5). Lp4CL2 and Lp4CL3 share 79% amino acid sequence identity, Lp4CL1 and Lp4CL2 have 61% amino acid sequence identity, while Lp4CL1 and Lp4CL3 have only 58% amino acid sequence identity. Regions of high sequence homology are more prevalent in the central and c-terminal regions of the enzyme. For example the sequence identity between amino acids 208 to 568 of each enzyme is 85% for Lp4CL2 and Lp4CL3, 72% for Lp4CL1 and Lp4CL2 and 67% for Lp4CL1 and Lp4CL3.

[0224]Lp4CL1, Lp4CL2 and Lp4CL3 share several common regions with other plant 4CLs. In particular, they contain the putative AMP-binding domain and the conserved GEICIRG motif, except for Lp4CL3 where the second isoleucine has been replaced with valine (FIG. 5). It has been proposed that domain II is associated with the catalytic activity of 4CL. Also, four Cys residues conserved in plant 4CLs are conserved in Lp4CL1, Lp4CL2 and Lp4CL3 (FIG. 5). These results suggest that the L. perenne cDNAs encode three divergent 4CL enzymes that are likely to have originated from three different 4CL genes.

Expression of Perennial Ryegrass 4CL Genes

[0225]Lp4CL1, Lp4CL2 and Lp4CL3 were used as hybridization probes in Northern blots with RNA prepared from different organs of perennial ryegrass at two developmental stages. All three probes hybridized to a single mRNA species of approximately 2.2-2.3 kb. Lp4CL1, Lp4CL2 and Lp4CL3 were expressed at both seedling and mature stages of development and in all organs tested. For Lp4CL2 and Lp4CL3 the strongest signal was found in RNA samples from seedling roots and mature stems (FIG. 6).

[0226]Lp4CL1, Lp4CL2 and Lp4CL3 were also used as hybridization probes in Northern blots with RNA prepared from tall fescue. All three probes hybridized to a similar mRNA species (2.3 kb) as that in perennial ryegrass (FIG. 6). The strongest signal was found in RNA samples from mature stems with weaker signals in RNA from roots and seedling shoots. No expression of Lp4CL1, Lp4CL2 or Lp4CL3 was observed in leaves. The three probes varied in their ability to hybridize to the corresponding homologues in tall fescue, with Lp4CL3 resulting in the highest signal and Lp4CL1 hybridizing only weakly.

[0227]To determine whether 4CL could be induced under stress conditions, leaves of perennial ryegrass seedlings were wounded. No increase in the transcript level upon wounding was observed with Lp4CL1, Lp4CL2 or Lp4CL3 (FIG. 7).

Genomic Organization of Perennial Ryegrass 4CL Genes

[0228]Perennial ryegrass DNA was digested with two restriction enzymes, HindIII or XbaI. Restriction sites for these enzymes are not present in the cDNA sequence of Lp4CL1, Lp4CL2 or Lp4CL3. When Lp4CL1, Lp4CL2 or Lp4CL3 was used as a probe, several DNA hybridizing fragments of varying intensity were revealed (FIG. 8). Each probe hybridized to a unique set of fragments, suggesting that Lp4CL1, Lp4CL2 and Lp4CL3 represent three different genes. Furthermore, Lp4CL1 and Lp4CL2 hybridized to 2 to 3 major fragments per digest which may represent either alleles of the same gene or indicate the presence of more than one gene in each class. The Lp4CL1, Lp4CL2 and Lp4CL3 probes also revealed several different size hybridizing DNA fragments in genomic Southern blots from tall fescue under high stringency conditions (FIG. 8), suggesting that three similar 4CL genes are present in F. arundinacea.

EXAMPLE 2 Starbucks
Isolation and Characterisation of a Cinnamoyl CoA Reductase (CCR) cDNA from Lolium perenne

[0229]A total of 500,000 phage were screened from a cDNA library constructed from ten-day-old etiolated L. perenne seedlings using a maize CCR probe. Ninety-three positive plaques were observed in the primary screen and five were subsequently analysed by restriction enzyme digestion. Four out of the five were identical. One of the four identical cDNAs, LpCCR1, was selected for further analysis (FIG. 9).

Nucleic Acid Sequence Analysis of Perennial Ryegrass CCR cDNA
Starbucks
[0230]The full nucleotide sequence of LpCCR1 was obtained and the amino acid sequence predicted (FIG. 10). LpCCR1 is a 1395 bp cDNA with 149 bp of 5' non-coding region and 160 bp of 3' non-coding region. An open reading frame of 1086 bp encodes a protein of 362 amino acids. The composition of the coding region was found to be 68% G+C rich. Codon usage was also examined and found to be biased towards XXC/G codons (94%), with XCG and XUA codons accounting for only 9% and 0.55% respectively. G+C richness and bias towards G and C in the third position of a codon triplet are previously reported characteristics of monocot genes.
Starbucks
Genomic Organization of Perennial Ryegrass CCR Gene
Starbucks
[0231]The number of CCR genes present in the ryegrass genome was determined by Southern blot analysis of genomic DNA from double haploid plants, using as probe a fragment of the LpCCR1 cDNA (LpCCR531, FIG. 9). Double haploid DNA reduces the complexity associated with allelic variation. Genomic DNA was cut with enzymes that do not cut the cDNA internally; DraI, BamHI, EcoRI, EcoRV, HindIII and XbaI, and the membrane was hybridised and washed under medium-stringency conditions. A single strongly hybridising band was evident in each lane (FIG. 11) indicating that there is a single copy of the LpCCR1 gene in the perennial ryegrass genome.

Expression of Perennial Ryegrass CCR Gene

[0232]To investigate the expression profile of the CCR gene in ryegrass, northern hybridisation analysis was carried out with total RNA extracted from roots and shoots at seedling growth stages (0.5-1 cm and 4-6 cm shoots) and roots, stem and leaves at mature growth stages (6 and 10 weeks). Seedlings were grown on filter paper in the dark at 25° C. and then transferred to soil and glasshouse conditions (25° C.) until the 6 and 10-week stages. Whole seedling total RNA from Festuca and Phalaris was included in the northern analysis. Hybridisation with LpCCR531 (FIG. 9) was performed at medium-stringency and the membrane was then washed at high-stringency. A transcript of approximately 1.5 kb was detected in all tissues, the level of expression varying with maturity and from one tissue type to another (FIG. 12). The LpCCR1 transcript appears to be more abundant in roots and stem than shoots and leaves. In the stem, transcript abundance increases from 6-weeks to 10-weeks; indicating that transcription in stem tissue is up-regulated as the plant matures. Expression was found predominantly in tissues such as stems and roots that are forming secondary cell walls indicating that LpCCR1 is constitutively involved in lignification.

EXAMPLE 3

Isolation and Characterisation of Cinnamyl Alcohol Dehydrogenase (CAD) cDNAs from Lolium perenne

target | December 4, 2009, 8:29am | #

mental health doctors
[0040]Preferably the fragment or variant includes a frame-shift mutation relative to Sequence ID No. 7.
The pedophile can be anyone
[0041]In a further preferred embodiment the present invention provides a fragment or variant of a nucleic acid selected from the group consisting of the nucleic acids with sequences shown in FIGS. 13, 14, 26 and 27 hereto (Sequence ID Nos: 9, 11, 14 and 16, respectively), wherein said fragment or variant is capable of modifying lignin biosynthesis in a plant via sense suppression of a gene encoding CAD in said plant.

[0042]Preferably, the fragment or variant includes a short deletion at or near the 3' or 5' end of a sequence selected from the group consisting of Sequence ID Nos. 9, 11, 14 and 16.mental health doctors
mental health doctors
[0043]Preferably, the fragment or variant includes including a frame-shift mutation relative to Sequence ID No. 9, 11, 14 or 16, remental health doctorsspectively.
mental health doctorsCrime / Punishment
[0044]In a further preferred embodiment, the present invention provides a fragment or variant of a nucleic acid selected from the group consisting of the nucleic acids with sequences shown in FIGS. 2, 3 and 4 hereto (Sequence ID Nos: 1, 3 and 5, respectively) wherein said fragment or variant is capable of modifying lignin biosynthesis in a plant via sense suppression of a gene encoding 4CL in said plant.
mental health doctors
[0045]Preferably, the fragment or variant includes a short deletion at or near the 3' or 5' end of a sequence selected from the group consisting of Sequence ID Nos: 1, 3 and 5.
mental health doctors
[0046]Preferably, the fragment or variant includes a frame-shift mutation relative to Sequence ID No: 1, 3 or 5, respectively.Crime / Punishment
mental health doctors
[0047]In a second aspect of the present invention there is provided a vector including a nucleic acid or nucleic acid fragment according to the present invention.
mental health doctors
[0048]In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment according to the present invention and a terminator; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked.
mental health doctorsCrime / Punishment
[0049]By "operatively linked" is meant that said regulatory element is capable of causing expression of said nucleic acid or nucleic acid fragment in a plant cell and said terminator is capable of terminating expression of said nucleic acid or nucleic acid fragment in a plant cell. Preferably, said regulatory element is upstream of said nucleic acid or nucleic acid fragment and said terminator is downstream of said nucleic acid or nucleic acid fragment.
mental health doctors
[0050]The vector may be of aCrime / PunishmentCrime / Punishmentny suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g.It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance publicderivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable or integrative or viable in the plant cell.
It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance public
[0051]The regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.
It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance public
[0052]Preferably the regulatory element is a promoter. A variety of promoters which may be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (e.g. monocotyledon or dicotyledon). Particularly suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, the rice Actin promoter, and ryegrass endogenous OMT, 4CL, CCR or CAD promoters.

[0053]A variety of terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.
DEA and IRS-CID began investigating
[0054]The vector, in addition to the regulatory element, the nucleic acid or nucleic acid fragment of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid or nucleic acid fragment, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.
DEA and IRS-CID began investigating
[0055]As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.
DEA and IRS-CID began investigating
[0056]Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to resultDefinitely not a child molester. in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance publiclinkers, for example including one or more restriction enzyme sites.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0057]The vectors of the present invention may be incorporated into a variety of plants, including monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turf grasses, corn, oat, sugarcane, wheat and barley), dicotyledons (such as arabidopsis, tobacco, legumes, alfalfa, oak, eucalyptus, maple, canola, soybean and chickpea) It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance publicand gymnosperms. In a preferred embodiment, the vectors are used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), more preferably perennial ryegrass (Lolium perenne) including forage and turf type cultivars.Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0058]Techniques for incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0059]Cells incorporating the vector of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0060]In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a vector of the present invention.

[0061]The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the plant cell, plant, plant seed or other plant part may be from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably a ryegrass, most preferably perennial ryegrass, including forage- and turf-type cultivars.

[0062]The present invention also provides a plant, plant seed or other plant part derived from a plant cell of the present invention.
It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance public
[0063]The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

[0064]In a further aspect of the present invention there is provided a method of modifying lignin biosynthesis in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment and/or a vector according to the present invention.
It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance public
[0065]By "an effective amount" is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by referIt is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance public
[0066]Using the methods and materials of the present invention, plant lignin biosynthesis may be increased, decreased or otherwise modified relative to an untransformed control plant. It may be increased or otherwise modified, for example, by incorporating additional copies of a sense nucleic acid or nucleic acid fragment of the present invention. It may be decreased, for example, by incorporating an antisense nucleic acid or nucleic acid fragment of the present invention or by incorporating a functionally active fragment or variant which is capable of modifying lignin biosynthesis in a plant via sense suppression. In addition, the number of copies of genes encoding for different enzymes in the lignin biosynthetic pathway may be manipulated to modify the relative amount of each monolignol synthesized, thereby leading to the formation of lignin having altered composition.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0067]Accordingly, in a preferred embodiment of this aspect of the invention there is provided a method of modifying lignin biosynthesis in a plant, said method including introducing into said plant in sense orientation an effective amount of a functionally active fragment or variant of a nucleic acid or nucleic acid fragment according to the present invention.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0068]Preferably the functionally active fragment or variant is capable of modifying lignin biosynthesis in the plant via sense suppression.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0069]Preferred functionally active fragments and variants for sense suppression include those hereinbefore described.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0070]In a still further aspect of the present invention there is provided use of a nucleic acid or nucleic acid fragment according to the present invention, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof, as a molecular genetic marker.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0071]More particularly, nucleic acids or nucleic acid fragments according to the present invention, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof, may be used as a molecular genetic marker for qualitative trait loci (QTL) tagging, mapping, DNA fingerprinting and in marker assisted selection, and may be used as candidate genes or perfect markers, particularly in ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention, and/or nucleotide sequence information thereof, may be used as molecular genetic markers in forage and turf grass improvement, e.g. tagging QTLs for dry matter digestibility, herbage quality, mechanical stress tolerance, disease resistance, insect pest resistance, plant stature and leaf and stem colour.

[0072]In a still further aspect of the present invention there is provided a substantially purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Fustuca) species, selected from the group consisting of the enzymes 4CL, CCR and CAD.

[0073]The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass L. perenne).

[0074]In a preferred embodiment of this aspect of the invention, the substantially purified or isolated enzyme 4CL includes an amino acid sequence selected from the group consisting of sequences shown in FIGS. 2, 3 and 4 hereto (Sequence ID Nos: 2, 4 and 6, respectively); and functionally active fragments and variants thereof.

[0075]In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated enzyme CCR includes an amino acid sequence selected from the group consisting of the sequence shown in FIG. 10 hereto (Sequence ID No: 8); and functionally active fragments and variants thereof.

[0076]In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated enzyme CAD includes an amino acid sequence selected from the group consisting of the sequence shown in FIGS. 13, 14, 26 and 27 hereto (Sequence ID Nos: 10, 12, 15 and 17, respectively); and functionally active fragments and variants thereof.

[0077]By "functionally active" in this context is meant that the fragment or variant has one or more of the biological properties of the enzymes 4CL, CCR and CAD, respectively. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the fragment or variant has at least approximately 60% identity to the relevant part of the above mentioned sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids.

[0078]In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid or nucleic acid fragment according to the present invention. Techniques for recombinantly producing polypeptides are well known to those skilled in the art.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0079]In a still further aspect of the present invention there is provided a lignin or modified lignin substantially or partially purified or isolated from a plant, plant seed or other plant part of the present invention.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0080]Such lignins may be modified from naturally occurring lignins in terms of the length, the degree of polymerisation (number of units), degree of branching and/or nature of linkagPedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,es between units.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0081]In a still further aspect, the present invention provides an isolated regulatory element capable of causing expression of an exogenous gene in plant cells. Preferably the regulatory element is isolated from a nucleic acid or nucleic acid fragment encoding OMT, 4CL, CCR or CAD.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0082]The regulatory element may be a nucleic acid molecule, including DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0083]Preferably the regulatory element includes a promoter, more preferably an O-methyltransferase promoter, even more preferably an O-methyltransferase promoter from a ryegrass (Lolium) or fescue (Festuca) species, more preferably a ryegrass, most preferably perennial ryegrass (Lolium perenne).Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0084]In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from the caffeic acid O-methyltransferase gene corresponding to the cDNA It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance publichomologue LpOMT1 from perennial ryegrass.
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0085]Preferably the regulatory element includes a nucleotide sequence including the first approximately 4630 nucleotides of the sequence shown in FIG. 18 hereto (Sequence ID No: 13); or a functionally active fragment or variant thereof.

[0086]By "functionally active" in this context is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of causing expression of a transgene in plant cells. It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance publicSuch variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the regulatory element. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. Preferably the fragment has a size of at least 100 nucleotides, more preferably at least 150 nucleotides, most preferably at least 200 nucleotides.

[0087]In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of:

[0088]Nucleotides--4581 to -1

[0089]Nucleotides--4285 to -1

[0090]Nucleotides--4020 to -1

[0091]Nucleotides--2754 to -1

[0092]Nucleotides--1810 to -1

[0093]Nucleotides--831 to -1

[0094]Nucleotides--560 to -1

[0095]Nucleotides--525 to -1

[0096]Nucleotides--274 to -1

[0097]Nucleotides--21 to -1

[0098]of FIG. 18 hereto (Sequence ID No: 13);

[0099]or a functionally active fragment or variant thereof.

[0100]In another preferred embodiment the regulatory element includes a 4 coumarate-CoA ligase promoter, even more preferably a 4 coumarate-CoA ligase promoter from a ryegrass (Lolium) or fescue (Festuca) species, more preferably a ryegrass, most preferably perennial ryegrass (Lolium perenne).

[0101]In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from the 4 coumarate-CoA ligase gene corresponding to the cDNA homologue Lp4CL2 from perennial ryegrass.

[0102]Preferably the regulatory element includes a nucleotide sequence including the first approximately 2206 nucleotides of the sequence shown in FIG. 38 hereto (Sequence ID No: 17); or a functionally active fragment or variant thereof.

[0103]By "functionally active" in this context is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of causing expression of a transgene in plant cells. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the regulatory element. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. Preferably the fragment has a size of at least 100 nucleotides, more preferably at least 150 nucleotides, most preferably at least 200 nucleotides.

[0104]In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of:

[0105]Nucleotides--2206 to -1

[0106]Nucleotides--1546 to -1

[0107]Nucleotides--1186 to -1

[0108]Nucleotides--406 to -1

[0109]Nucleotides--166 to -1

[0110]of FIG. 38 hereto (Sequence ID No: 17);

[0111]or a functionally active fragment or variant thereof.

[0112]In another preferred embodiment the regulatory element includes a cinnamoyl-CoA reductase promoter, even more preferably a cinnamoyl-CoA reductase promoter from a ryegrass (Lolium) or fescue (Festuca) species, more preferably a ryegrass, most preferably perennial ryegrass (Lolium perenne).

[0113]In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from the cinnamoyl-CoA reductase gene corresponding to the LpCCR1 cDNA from perennial ryegrass.

[0114]Preferably the regulatory element includes a nucleotide sequence including the first approximately 6735 nucleotides of the sequence shown in FIG. 39 hereto (Sequence ID No: 18); or a functionally active fragment or variant thereof.
Starbucks
[0115]By "functionally active" in this context is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of causing expression of a transgene in plant cells. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the regulatory element. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. Preferably the fragment has a size of at least 100 nucleotides, more preferably at least 150 nucleotides, most preferably at least 200 nucleotides.
Starbucks
[0116]In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of:
Starbucks
[0117]Nucleotides--6735 to -1
It is the mission of the LAPD to safeguard the lives and property of the people we serve. To reduce the incidence and fear of crime, and to enhance public
[0118]Nucleotides--5955 to -1
Starbucks
[0119]Nucleotides--5415 to -1

[0120]Nucleotides--4455 to -1

[0121]Nucleotides--4035 to -1
DEA and IRS-CID began investigating
[0122]Nucleotides--3195 to -1
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0123]Nucleotides--2595 to -1

[0124]Nucleotides--1755 to -1

[0125]Nucleotides--1275 to -1

[0126]Nucleotides--495 to -1
Pedophiles can be anyone -- old or young, rich or poor, educated or uneducated, non-professional or professional, and of any race. However,
[0127]Nucleotides--255 to -1
DEA and IRS-CID began investigating
[0128]Nucleotides--75 to -1

[0129]of FIG. 39 hereto (Sequence ID No: 18);

[0130]or a functionally active fragment or variant thereof.
DEA and IRS-CID began investigating
[0131]By an "exogenous gene" is meant a gene not natively linked to said regulatory element. In certain embodiments of the present invention the exogenous gene is also not natively found in the relevant plant or plant cell.
DEA and IRS-CID began investigating
[0132]The exogenous gene may be of any suitable type. The exogenous gene may be a nucleic acid such as DNA (e.g. cDNA or genomic DNA) or RNA (e.g. mRNA), and combinations thereof. The exogenous gene may correspond to a target gene, for example a gene capable of influencing disease resistance, herbage digestibility, nutrient quality, mineral content or drought tolerance or be a fragment or variant (such as an analogue, derivative or mutant) thereof which is capable of modifying expression of said target gene. Such variants include nucleic acid sequences which are antisense to said target gene or an analogue, derivative, mutant or fragment thereof.557PRTLolium perenne 6Met Gly Ser Val Pro Glu Glu Ser Val Val Ala Val Ala Pro Ala Glu1 5 10 15Thr Val Phe Arg Ser Lys Leu Pro Asp Ile Glu Ile AMet Asn Leu Leu Arg Asn Cys Pro Glu 85 90 95Phe Ala Phe Ser Phe Leu Gly Ala Ala Arg Leu Gly Ala Ala Thr Thr 100 105 110Thr Ala Asn Pro Phe Tyr Thr Pro The transgene may code for a protein or RNA sequence depending the target condition and whether down or up-regulation of gene expression is required. Preferably, the target gene is selected from exogenous coding sequences coding for mRNA for a protein, this protein may be of bacterial origin (such as enzymes involved in cell wall modification and cell wall metabolism, cytokinin biosynthesis), or eukaryotic origin (such as pharmaceutically active polypeptides) or of plant origin (such as enzymes involved in the synthesis of phenolic compounds, cell wall metabolism, sugar metabolism, lignin biosynthesis). Preferably, the target gene is selected from the group comprising O-methyltransferase, 4 coumarate CoA-ligase, cinnamoyl CoA reductase, cinnamyl alcohol dehydrogenase, cinnamate 4 hydroxylase, phenolase, laccase, peroxidase, coniferol glucosyl transferase, coniferin beta-glucosidase, phenylalanine ammonia lyase, ferulate 5-hydroxylase, chitinase, glucanase, isopentenyltransferase, xylanase.

SuicideBridgePasadena | December 4, 2009, 9:08am | #

[0133]The plant cells, in which the regulatory element of the present invention is capable of causing expression of an exogenous gene, may be of any suitable type. The plant cells may be from monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turf grasses, corn, grains, oat, sugarcane, wheat and barley), dicotyledons (such as arabidopsis, tobacco, legumes, alfalfa, oak, eucalyptus and maple) and gymnosperms. Preferably the plant cells are from a monocotyledon, more preferably a grass species such as a ryegrass (Lolium) or fescue (Festuca) species, even more preferably a ryegrass, most preferably perennial ryegrass (Lolium perenne).

[0134]The regulatory element according to the present invention may be used to express exogenous genes to which it is operatively linked in the production of transgenic plants.

[0135]Accordingly, in a further aspect of the present invention there is provided a vector including a regulatory element according to the present invention.

[0136]In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element according to the present invention, an exogenous gene as hereinbefore described, and a terminator; said regulatory element, exogenous gene and terminator being operatively linked, such that said regulatory element is capable of causing expression of said exogenous gene in plant cells. Preferably, said regulatory element is upstream of said exogenous gene and said terminator is downstream of said exogenous gene.

[0137]The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable on integrative or viable in the plant cell.
Starbucks
[0138]The terminator may be of any suitable type and includes for example polyadenylation signals, such as the Cauliflower Mosaic Virus 35S polyA (CaMV 35S polyA) and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.

[0139]The vector, in add Starbucksition to the regulatory element, the exogenous nucleic acid and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or Starbuckspat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.
Starbucks
[0140]The regulatory element of the present invention may also be used with other full promoters or partial promoter elements. Starbucks

[0141]As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.

[0142]Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said transgene. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction sites.

[0143]The vectors of the present invention may be incorporated into a variety of plants, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the vectors are used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), more preferably perennial ryegrass (Lolium perenne) including forage- and turf-type cultivars.

[0144]Techniques for incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.

[0145]Cells incorporating the vector of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

[0146]In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a vector of the present invention.

[0147]The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the plant cell, plant, plant seed or other plant part is from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably perennial ryegrass (Lolium perenne), including forage- and turf-type cultivars.
Starbucks
[0148]The present invention also provides a plant, plant seed, or other plant part derived from a plant cell of the present invention.

[0149]The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

[0150]In a still further aspect of the present invention there is provided a recombinant plant genome including a regulatory element according to the present invention.

[0151]In a preferred embodiment of this aspect of the invention the recombinant plant genome further includes an exogenous gene operatively linked to said regulatory element.
Starbucks
[0152]In a further aspect of the present invention there is provided a method for expressing an exogenous gene in plant cells, said method including introducing into said plant cells an effective amount of a regulatory element and/or a vector according to the present invention.

[0153]By "an effective amount" is meant an amount sufficient to result in an identifiable phenotypic change in said plant cells or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant cell, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

[0154]The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

IN THE FIGURES

[0155]FIG. 1 shows plasmid maps of the three cDNAs encoding perennial ryegrass 4CL homologues.

[0156]FIG. 2 shows the nucleotide (Sequence ID No: 1) and amino acid (Sequence ID No: 2) sequences of Lp4CL1.

[0157]FIG. 3 shows the nucleotide (Sequence ID No: 3) and amino acid (Sequence ID No: 4) sequences of Lp4CL2.

[0158]FIG. 4 shows the nucleotide (Sequence ID No: 5) and amino acid (Sequence ID No: 6) sequences of Lp4CL3.

[0159]FIG. 5 shows amino acid sequence alignment of deduced proteins encoded by Lp4CL1 (Sequence ID No: 2), Lp4CL2 (Sequence ID No: 4) and Lp4CL3 (Sequence ID No: 6).

[0160]FIG. 6 shows northern hybridisation analysis of developing perennial ryegrass using Lp4CL1, Lp4CL2 and Lp4CL3 as hybridisation probes. SR: roots from seedlings (3-5 d post-germination), SS: shoots from seedlings (3-5 d post-germination), ML: leaves from 12-week-old plants, MS: stems from 12-week-old plants. Blots were washed in 0.2×SSPE, 0.1% SDS at 65° C. Lp4CL1, Lp4CL2 and Lp4CL3 do not cross hybridise at this stringency. Sizes are given in kb.

[0161]FIG. 7 shows northern hybridisation analysis showing the time course of expression of 4CL mRNA in wounded perennial ryegrass leaves. Sizes are given in kb.

[0162]FIG. 8 shows genomic Southern hybridisation analysis using Lp4CL1, Lp4CL2 and Lp4CL3 as hybridisation probes. 10 μg of digested perennial ryegrass genomic DNA or 20 μg of digested tall fescue genomic DNA were separated on a 1.0% agarose gel, transferred to Hybond N.sup.+ membranes and then hybridised with 32P labelled Lp4CL1, Lp4CL2 or Lp4CL3 probes. The ryegrass Lp4CL1, Lp4CL2 and Lp4CL3 genes reveal homologous sequences in tall fescue and indicate that the ryegrass 4CL genes can be used to isolate and to manipulate the expression of the tall fescue (Festuca arundinacea) 4CL genes.

[0163]FIG. 9 shows restriction map of LpCCR1. An L. perenne seedling cDNA library constructed in Uni-ZAP® (Stratagene) was screened in a solution containing 10×PIPES, 50% deionised formamide and 10% SDS at 42° C. Filters were washed at room temperature, three times in 0.1% SDS, 2×SSPE and then twice in 0.1% SDS, 0.2×SSPE. The location of the probe used for northern and Southern hybridisation analyses is indicated by the black line labelled LpCCR531.

[0164]FIG. 10 shows the nucleotide (Sequence ID No: 7) and amino acid (Sequence ID No: 8) sequences of LpCCR1.

[0165]FIG. 11 shows Southern hybridisation analysis of DNA from double haploid (DH) perennial ryegrass using LpCCR1 as hybridisation probe. 10 μg of DH genomic DNA was digested with DraI, BamHI, EcoRI, EcoRV, HindIII or XbaI, separated on a 1% agarose gel and then capillary blotted onto nylon membrane (Amersham Hybond-N). The membrane was probed with the digoxigenin (DIG) labelled LpCCR531 fragment at 25 ng/ml in the hybridisation solution. Hybridisation was in 4×SSC, 50% formamide, 0.1% N-Lauroyl-sarcosine, 0.02% SDS, 2% Blocking solution at 42° C. The membrane was washed twice for five minutes in 2×SSC, 0.1% SDS at room temperature, then twice for fifteen minutes in 0.5×SSC, 0.1% SDS at 68° C. Molecular weight was determined by comparison to a DIG-labelled marker (Roche Molecular Biochemicals).

[0166]FIG. 12 shows northern hybridisation analysis of RNA samples from different organs and developmental stages of perennial ryegrass using LpCCR1 probe. Roots from seedlings (3-5 d post-germination), shoots from seedlings (3-5 d post-germination), roots from seedlings (7-10 d post-germination), leaves from seedlings (7-10 d post-germination), roots from 6 and 10 week old plants, leaves from 6 and 10 week old plants, stems from 6 and 10 week old plants, whole seedling from 11 day old Phalaris and 7 day old Festuca.

[0167]Total RNA was isolated using Trizol (GibcoBRL) and 15 μg was separated on a 1.2% Agarose gel containing 6% formamide and then capillary blotted onto nylon membrane (Amersham Hybond-N). The membrane was stained with 0.2% methylene blue/0.3M sodium acetate to visualise the marker and ensure that RNA was evenly loaded. 50 ng LpCCR531 was random-labelled with 32P-dCTP (Amersham Megaprime) and hybridisation conditions were 4×SSC, 50% formamide, 0.5% SDS, 5×denhardt solution, 5% dextrane sulphate, 0.1% Herring

[0178]FIG. 23 shows the isolation of an Lp4CL genomic clone. A) Southern hybridisation analysis of 4CL genomic clone λLp4CL2 digested with BamHI, KpnIs or SalI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled 4CL1 hybridisation probe. B) 10 μl of a standard PCR reaction using forward and reverse oligonucleotides designed to positions outlined on C). The PCR products were separated on a 0.8% agarose gel and stained with ethidium bromide. C) Map showing the genomic gene organisation of λLp4CL2 based on sequence and PCR results.

[0179]FIG. 24 shows the isolation of an Lp4CL genomic clone. A) Southern hybridisation analysis of 4CL genomic clone λLp4CL2 digested with BamHI, KpnI, SalI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled 4CL1 probe. B) Map showing the genomic gene organisation of Lp4CL2 clone 1 and the promoter region of clone 2.

[0180]FIG. 25 shows plasmid map of plant transformation vector carrying the gusA gene under control of the perennial ryegrass Lp4CL2 promoter (Lp4CL2::gusA).

[0181]FIG. 26 shows nucleotide (Sequence ID No: 14) and amino acid (Sequence ID No: 15) sequences of genomic clone CAD2 cv Barlano (Intron 1 and first 111 bp of the coding region are missing).

[0182]FIG. 27 shows nucleotide (Sequence ID No: 16) and amino acid (Sequence ID No: 15) sequences of coding sequence deduced from genomic clone CAD2 cv Barlano (region in bold is missing from the genomic clone).

[0183]FIG. 28 shows the isolation of LpCAD2 genomic clone. A) Southern hybridization analysis of CAD genomic clone λLpCAD2 digested with BamHI, EcoRI, KpnI, SalI or XbaI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled CAD2 hybridisation probe. B) Map showing the genomic gene organisation of λLpCAD2 based on sequence results.

[0184]FIG. 29 shows A) Sense and antisense Lp4CL1, Lp4CL2 and Lp4CL3 transformation vectors under control of the CaMV 35S promoter; B) Sense and antisense Lp4CL1, Lp4CL2 and Lp4CL3 transformation vectors under control of the maize ubiquitin promoter.










[0133]The plant cells, in which the regulatory element of the present invention is capable of causing expression of an exogenous gene, may be of any suitable type. The plant cells may be from monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turf grasses, corn, grains, oat, sugarcane, wheat and barley), dicotyledons (such as arabidopsis, tobacco, legumes, alfalfa, oak, eucalyptus and maple) and gymnosperms. Preferably the plant cells are from a monocotyledon, more preferably a grass species such as a ryegrass (Lolium) or fescue (Festuca) species, even more preferably a ryegrass, most preferably perennial ryegrass (Lolium perenne).

[0134]The regulatory element according to the present invention may be used to express exogenous genes to which it is operatively linked in the production of transgenic plants.

[0135]Accordingly, in a further aspect of the present invention there is provided a vector including a regulatory element according to the present invention.

[0136]In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element according to the present invention, an exogenous gene as hereinbefore described, and a terminator; said regulatory element, exogenous gene and terminator being operatively linked, such that said regulatory element is capable of causing expression of said exogenous gene in plant cells. Preferably, said regulatory element is upstream of said exogenous gene and said terminator is downstream of said exogenous gene.

[0137]The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable on integrative or viable in the plant cell.
Starbucks
[0138]The terminator may be of any suitable type and includes for example polyadenylation signals, such as the Cauliflower Mosaic Virus 35S polyA (CaMV 35S polyA) and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.

[0139]The vector, in add Starbucksition to the regulatory element, the exogenous nucleic acid and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or Starbuckspat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.
Starbucks
[0140]The regulatory element of the present invention may also be used with other full promoters or partial promoter elements. Starbucks

[0141]As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.

[0142]Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said transgene. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction sites.

[0143]The vectors of the present invention may be incorporated into a variety of plants, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the vectors are used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), more preferably perennial ryegrass (Lolium perenne) including forage- and turf-type cultivars.

[0144]Techniques for incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.

[0145]Cells incorporating the vector of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

[0146]In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a vector of the present invention.

[0147]The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the plant cell, plant, plant seed or other plant part is from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably perennial ryegrass (Lolium perenne), including forage- and turf-type cultivars.
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[0148]The present invention also provides a plant, plant seed, or other plant part derived from a plant cell of the present invention.

[0149]The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

[0150]In a still further aspect of the present invention there is provided a recombinant plant genome including a regulatory element according to the present invention.

[0151]In a preferred embodiment of this aspect of the invention the recombinant plant genome further includes an exogenous gene operatively linked to said regulatory element.
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[0152]In a further aspect of the present invention there is provided a method for expressing an exogenous gene in plant cells, said method including introducing into said plant cells an effective amount of a regulatory element and/or a vector according to the present invention.

[0153]By "an effective amount" is meant an amount sufficient to result in an identifiable phenotypic change in said plant cells or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant cell, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

[0154]The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

IN THE FIGURES

[0155]FIG. 1 shows plasmid maps of the three cDNAs encoding perennial ryegrass 4CL homologues.

[0156]FIG. 2 shows the nucleotide (Sequence ID No: 1) and amino acid (Sequence ID No: 2) sequences of Lp4CL1.

[0157]FIG. 3 shows the nucleotide (Sequence ID No: 3) and amino acid (Sequence ID No: 4) sequences of Lp4CL2.

[0158]FIG. 4 shows the nucleotide (Sequence ID No: 5) and amino acid (Sequence ID No: 6) sequences of Lp4CL3.

[0159]FIG. 5 shows amino acid sequence alignment of deduced proteins encoded by Lp4CL1 (Sequence ID No: 2), Lp4CL2 (Sequence ID No: 4) and Lp4CL3 (Sequence ID No: 6).

[0160]FIG. 6 shows northern hybridisation analysis of developing perennial ryegrass using Lp4CL1, Lp4CL2 and Lp4CL3 as hybridisation probes. SR: roots from seedlings (3-5 d post-germination), SS: shoots from seedlings (3-5 d post-germination), ML: leaves from 12-week-old plants, MS: stems from 12-week-old plants. Blots were washed in 0.2×SSPE, 0.1% SDS at 65° C. Lp4CL1, Lp4CL2 and Lp4CL3 do not cross hybridise at this stringency. Sizes are given in kb.

[0161]FIG. 7 shows northern hybridisation analysis showing the time course of expression of 4CL mRNA in wounded perennial ryegrass leaves. Sizes are given in kb.

[0162]FIG. 8 shows genomic Southern hybridisation analysis using Lp4CL1, Lp4CL2 and Lp4CL3 as hybridisation probes. 10 μg of digested perennial ryegrass genomic DNA or 20 μg of digested tall fescue genomic DNA were separated on a 1.0% agarose gel, transferred to Hybond N.sup.+ membranes and then hybridised with 32P labelled Lp4CL1, Lp4CL2 or Lp4CL3 probes. The ryegrass Lp4CL1, Lp4CL2 and Lp4CL3 genes reveal homologous sequences in tall fescue and indicate that the ryegrass 4CL genes can be used to isolate and to manipulate the expression of the tall fescue (Festuca arundinacea) 4CL genes.

[0163]FIG. 9 shows restriction map of LpCCR1. An L. perenne seedling cDNA library constructed in Uni-ZAP® (Stratagene) was screened in a solution containing 10×PIPES, 50% deionised formamide and 10% SDS at 42° C. Filters were washed at room temperature, three times in 0.1% SDS, 2×SSPE and then twice in 0.1% SDS, 0.2×SSPE. The location of the probe used for northern and Southern hybridisation analyses is indicated by the black line labelled LpCCR531.

[0164]FIG. 10 shows the nucleotide (Sequence ID No: 7) and amino acid (Sequence ID No: 8) sequences of LpCCR1.

[0165]FIG. 11 shows Southern hybridisation analysis of DNA from double haploid (DH) perennial ryegrass using LpCCR1 as hybridisation probe. 10 μg of DH genomic DNA was digested with DraI, BamHI, EcoRI, EcoRV, HindIII or XbaI, separated on a 1% agarose gel and then capillary blotted onto nylon membrane (Amersham Hybond-N). The membrane was probed with the digoxigenin (DIG) labelled LpCCR531 fragment at 25 ng/ml in the hybridisation solution. Hybridisation was in 4×SSC, 50% formamide, 0.1% N-Lauroyl-sarcosine, 0.02% SDS, 2% Blocking solution at 42° C. The membrane was washed twice for five minutes in 2×SSC, 0.1% SDS at room temperature, then twice for fifteen minutes in 0.5×SSC, 0.1% SDS at 68° C. Molecular weight was determined by comparison to a DIG-labelled marker (Roche Molecular Biochemicals).

[0166]FIG. 12 shows northern hybridisation analysis of RNA samples from different organs and developmental stages of perennial ryegrass using LpCCR1 probe. Roots from seedlings (3-5 d post-germination), shoots from seedlings (3-5 d post-germination), roots from seedlings (7-10 d post-germination), leaves from seedlings (7-10 d post-germination), roots from 6 and 10 week old plants, leaves from 6 and 10 week old plants, stems from 6 and 10 week old plants, whole seedling from 11 day old Phalaris and 7 day old Festuca.

[0167]Total RNA was isolated using Trizol (GibcoBRL) and 15 μg was separated on a 1.2% Agarose gel containing 6% formamide and then capillary blotted onto nylon membrane (Amersham Hybond-N). The membrane was stained with 0.2% methylene blue/0.3M sodium acetate to visualise the marker and ensure that RNA was evenly loaded. 50 ng LpCCR531 was random-labelled with 32P-dCTP (Amersham Megaprime) and hybridisation conditions were 4×SSC, 50% formamide, 0.5% SDS, 5×denhardt solution, 5% dextrane sulphate, 0.1% Herring

[0178]FIG. 23 shows the isolation of an Lp4CL genomic clone. A) Southern hybridisation analysis of 4CL genomic clone λLp4CL2 digested with BamHI, KpnIs or SalI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled 4CL1 hybridisation probe. B) 10 μl of a standard PCR reaction using forward and reverse oligonucleotides designed to positions outlined on C). The PCR products were separated on a 0.8% agarose gel and stained with ethidium bromide. C) Map showing the genomic gene organisation of λLp4CL2 based on sequence and PCR results.

[0179]FIG. 24 shows the isolation of an Lp4CL genomic clone. A) Southern hybridisation analysis of 4CL genomic clone λLp4CL2 digested with BamHI, KpnI, SalI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled 4CL1 probe. B) Map showing the genomic gene organisation of Lp4CL2 clone 1 and the promoter region of clone 2.

[0180]FIG. 25 shows plasmid map of plant transformation vector carrying the gusA gene under control of the perennial ryegrass Lp4CL2 promoter (Lp4CL2::gusA).

[0181]FIG. 26 shows nucleotide (Sequence ID No: 14) and amino acid (Sequence ID No: 15) sequences of genomic clone CAD2 cv Barlano (Intron 1 and first 111 bp of the coding region are missing).

[0182]FIG. 27 shows nucleotide (Sequence ID No: 16) and amino acid (Sequence ID No: 15) sequences of coding sequence deduced from genomic clone CAD2 cv Barlano (region in bold is missing from the genomic clone).

[0183]FIG. 28 shows the isolation of LpCAD2 genomic clone. A) Southern hybridization analysis of CAD genomic clone λLpCAD2 digested with BamHI, EcoRI, KpnI, SalI or XbaI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled CAD2 hybridisation probe. B) Map showing the genomic gene organisation of λLpCAD2 based on sequence results.

[0184]FIG. 29 shows A) Sense and antisense Lp4CL1, Lp4CL2 and Lp4CL3 transformation vectors under control of the CaMV 35S promoter; B) Sense and antisense Lp4CL1, Lp4CL2 and Lp4CL3 transformation vectors under control of the maize ubiquitin promoter.
[0137]The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable on integrative or viable in the plant cell.
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[0138]The terminator may be of any suitable type and includes for example polyadenylation signals, such as the Cauliflower Mosaic Virus 35S polyA (CaMV 35S polyA) and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.

[0139]The vector, in add Starbucksition to the regulatory element, the exogenous nucleic acid and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or Starbuckspat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.
Starbucks
[0140]The regulatory element of the present invention may also be used with other full promoters or partial promoter elements. Starbucks

[0141]As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.

[0142]Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said transgene. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction sites.

[0143]The vectors of the present invention may be incorporated into a variety of plants, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the vectors are used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), more preferably perennial ryegrass (Lolium perenne) including forage- and turf-type cultivars.

[0144]Techniques for incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.

[0145]Cells incorporating the vector of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

[0146]In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a vector of the present invention.

[0147]The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the plant cell, plant, plant seed or other plant part is from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably perennial ryegrass (Lolium perenne), including forage- and turf-type cultivars.
Starbucks
[0148]The present invention also provides a plant, plant seed, or other plant part derived from a plant cell of the present invention.

[0149]The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

[0150]In a still further aspect of the present invention there is provided a recombinant plant genome including a regulatory element according to the present invention.

[0151]In a preferred embodiment of this aspect of the invention the recombinant plant genome further includes an exogenous gene operatively linked to said regulatory element.
Starbucks
[0152]In a further aspect of the present invention there is provided a method for expressing an exogenous gene in plant cells, said method including introducing into said plant cells an effective amount of a regulatory element and/or a vector according to the present invention.

[0153]By "an effective amount" is meant an amount sufficient to result in an identifiable phenotypic change in said plant cells or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant cell, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

[0154]The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

IN THE FIGURES

[0155]FIG. 1 shows plasmid maps of the three cDNAs encoding perennial ryegrass 4CL homologues.

[0156]FIG. 2 shows the nucleotide (Sequence ID No: 1) and amino acid (Sequence ID No: 2) sequences of Lp4CL1.

[0157]FIG. 3 shows the nucleotide (Sequence ID No: 3) and amino acid (Sequence ID No: 4) sequences of Lp4CL2.

[0158]FIG. 4 shows the nucleotide (Sequence ID No: 5) and amino acid (Sequence ID No: 6) sequences of Lp4CL3.

[0159]FIG. 5 shows amino acid sequence alignment of deduced proteins encoded by Lp4CL1 (Sequence ID No: 2), Lp4CL2 (Sequence ID No: 4) and Lp4CL3 (Sequence ID No: 6).

[0160]FIG. 6 shows northern hybridisation analysis of developing perennial ryegrass using Lp4CL1, Lp4CL2 and Lp4CL3 as hybridisation probes. SR: roots from seedlings (3-5 d post-germination), SS: shoots from seedlings (3-5 d post-germination), ML: leaves from 12-week-old plants, MS: stems from 12-week-old plants. Blots were washed in 0.2×SSPE, 0.1% SDS at 65° C. Lp4CL1, Lp4CL2 and Lp4CL3 do not cross hybridise at this stringency. Sizes are given in kb.

[0161]FIG. 7 shows northern hybridisation analysis showing the time course of expression of 4CL mRNA in wounded perennial ryegrass leaves. Sizes are given in kb.

[0162]FIG. 8 shows genomic Southern hybridisation analysis using Lp4CL1, Lp4CL2 and Lp4CL3 as hybridisation probes. 10 μg of digested perennial ryegrass genomic DNA or 20 μg of digested tall fescue genomic DNA were separated on a 1.0% agarose gel, transferred to Hybond N.sup.+ membranes and then hybridised with 32P labelled Lp4CL1, Lp4CL2 or Lp4CL3 probes. The ryegrass Lp4CL1, Lp4CL2 and Lp4CL3 genes reveal homologous sequences in tall fescue and indicate that the ryegrass 4CL genes can be used to isolate and to manipulate the expression of the tall fescue (Festuca arundinacea) 4CL genes.

[0163]FIG. 9 shows restriction map of LpCCR1. An L. perenne seedling cDNA library constructed in Uni-ZAP® (Stratagene) was screened in a solution containing 10×PIPES, 50% deionised formamide and 10% SDS at 42° C. Filters were washed at room temperature, three times in 0.1% SDS, 2×SSPE and then twice in 0.1% SDS, 0.2×SSPE. The location of the probe used for northern and Southern hybridisation analyses is indicated by the black line labelled LpCCR531.

[0164]FIG. 10 shows the nucleotide (Sequence ID No: 7) and amino acid (Sequence ID No: 8) sequences of LpCCR1.

[0165]FIG. 11 shows Southern hybridisation analysis of DNA from double haploid (DH) perennial ryegrass using LpCCR1 as hybridisation probe. 10 μg of DH genomic DNA was digested with DraI, BamHI, EcoRI, EcoRV, HindIII or XbaI, separated on a 1% agarose gel and then capillary blotted onto nylon membrane (Amersham Hybond-N). The membrane was probed with the digoxigenin (DIG) labelled LpCCR531 fragment at 25 ng/ml in the hybridisation solution. Hybridisation was in 4×SSC, 50% formamide, 0.1% N-Lauroyl-sarcosine, 0.02% SDS, 2% Blocking solution at 42° C. The membrane was washed twice for five minutes in 2×SSC, 0.1% SDS at room temperature, then twice for fifteen minutes in 0.5×SSC, 0.1% SDS at 68° C. Molecular weight was determined by comparison to a DIG-labelled marker (Roche Molecular Biochemicals).

[0166]FIG. 12 shows northern hybridisation analysis of RNA samples from different organs and developmental stages of perennial ryegrass using LpCCR1 probe. Roots from seedlings (3-5 d post-germination), shoots from seedlings (3-5 d post-germination), roots from seedlings (7-10 d post-germination), leaves from seedlings (7-10 d post-germination), roots from 6 and 10 week old plants, leaves from 6 and 10 week old plants, stems from 6 and 10 week old plants, whole seedling from 11 day old Phalaris and 7 day old Festuca.

[0167]Total RNA was isolated using Trizol (GibcoBRL) and 15 μg was separated on a 1.2% Agarose gel containing 6% formamide and then capillary blotted onto nylon membrane (Amersham Hybond-N). The membrane was stained with 0.2% methylene blue/0.3M sodium acetate to visualise the marker and ensure that RNA was evenly loaded. 50 ng LpCCR531 was random-labelled with 32P-dCTP (Amersham Megaprime) and hybridisation conditions were 4×SSC, 50% formamide, 0.5% SDS, 5×denhardt solution, 5% dextrane sulphate, 0.1% Herring

[0178]FIG. 23 shows the isolation of an Lp4CL genomic clone. A) Southern hybridisation analysis of 4CL genomic clone λLp4CL2 digested with BamHI, KpnIs or SalI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled 4CL1 hybridisation probe. B) 10 μl of a standard PCR reaction using forward and reverse oligonucleotides designed to positions outlined on C). The PCR products were separated on a 0.8% agarose gel and stained with ethidium bromide. C) Map showing the genomic gene organisation of λLp4CL2 based on sequence and PCR results.

[0179]FIG. 24 shows the isolation of an Lp4CL genomic clone. A) Southern hybridisation analysis of 4CL genomic clone λLp4CL2 digested with BamHI, KpnI, SalI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled 4CL1 probe. B) Map showing the genomic gene organisation of Lp4CL2 clone 1 and the promoter region of clone 2.

[0180]FIG. 25 shows plasmid map of plant transformation vector carrying the gusA gene under control of the perennial ryegrass Lp4CL2 promoter (Lp4CL2::gusA).

[0181]FIG. 26 shows nucleotide (Sequence ID No: 14) and amino acid (Sequence ID No: 15) sequences of genomic clone CAD2 cv Barlano (Intron 1 and first 111 bp of the coding region are missing).

[0182]FIG. 27 shows nucleotide (Sequence ID No: 16) and amino acid (Sequence ID No: 15) sequences of coding sequence deduced from genomic clone CAD2 cv Barlano (region in bold is missing from the genomic clone).

[0183]FIG. 28 shows the isolation of LpCAD2 genomic clone. A) Southern hybridization analysis of CAD genomic clone λLpCAD2 digested with BamHI, EcoRI, KpnI, SalI or XbaI. DNA was separated on a 0.8% agarose gel, transferred to Hybond N and hybridized with a DIG labelled CAD2 hybridisation probe. B) Map showing the genomic gene organisation of λLpCAD2 based on sequence results.

[0184]FIG. 29 shows A) Sense and antisense Lp4CL1, Lp4CL2 and Lp4CL3 transformation vectors under control of the CaMV 35S promoter; B) Sense and antisense Lp4CL1, Lp4CL2 and Lp4CL3 transformation vectors under control of the maize ubiquitin promoter.

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