Dna Recombinant Technology And Gene Silencing In Plants Biology Essay

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Recombinant DNA, also known as in vitro recombination, is a technique involved in creating and purifying desired genes. Molecular cloning (i.e. gene cloning) involves creating recombinant DNA and introducing it into a host cell to be replicated. One of the basic strategies of molecular cloning is to move desired genes from a large, complex genome to a small, simple one. The process of in vitro recombination makes it possible to cut different strands of DNA, in vitro (outside the cell), with a restriction enzyme and join the DNA molecules together via complementary base pairing.

Homology-dependent gene silencing has been discovered in various organisms, ranging from plants to animals to fungi. Several terms have come out because of using different experimental system to study this phenomenon, often describing potentially similar processes. Silencing is generally based on two properties; the inducing agent and the mechanism of silencing. The terms are:

Post-transcriptional gene silencing (PTGS)

This is a general term that applies to RNA interference (RNAi) in animals, and to some types of virally- and transgene-induced silencing in plants. The transcription of the gene is unaffected; however, gene expression is lost because mRNA molecules become unstable.

Transcriptional gene silencing (TGS)

This is generally observed in plants but has also been seen in animals. Gene expression is reduced by a blockade at the transcriptional level. Evidence indicates that transcriptional suppression might be caused by chromatin modification or DNA methylation.

Transgene-induced silencing

Silencing is caused by the presence of transgenes in the genome. Suppression is usually related to copy number. Tandemly arrayed transgenes are more effective inducers of silencing than dispersed transgenes, with inverted repeats being the most effective. Silencing can occur transcriptionally or post-transcriptionally.

Virus-induced silencing

Silencing is induced by the presence of viral genomic RNA. Only replication-competent viruses cause silencing, indicating that double-stranded RNA (dsRNA) molecules might be the inducing agents. In virus-infected plants, cytoplasmic siRNA silencing, which is an anti-viral defense response, is initiated by dsRNA that could be viral replication intermediates or 'aberrant' RNAs, or single-stranded RNAs (ssRNA) that become dsRNA by host-encoded RNA-dependent RNA polymerase (RdRP) (Dalmay, 2000).

Plant viruses are important pathogens that are poorly controlled and whose molecular basis for disease development is still under investigation. The majority of plant-infecting viruses have RNA genomes, except caulimoviruses, nanoviruses and geminiviruses. Caulimoviruses have a dsDNA genome that replicates through an RNA intermediate using reverse transcription (Hul, 1987). Geminiviruses and nanoviruses have ssDNA genomes and replicate through a dsDNA intermediate by a rolling circle mechanism (Laufs, 1995).

Cosuppression

It is silencing of an endogenous gene due to the presence of a homologous transgene or virus. Cosuppression can occur at the transcriptional or post-transcriptional level (PTGS or TGS).

RNAi

This type of PTGS is induced directly by dsRNA. It was first defined in Caenorhabditis elegans and seems to be mechanistically related, if not identical, to PTGS in plants. Long double-stranded RNA (dsRNA) into short-interfering (21-26 nucleotides) RNAs (Hamilton, 1999) by a Ribonuclease III-like enzyme termed DICER (Bernstein, 2001). In plants, four Dicer-like (DCL) enzymes have been identified in Arabidopsis thaliana (Schauer, 2002); however, distinct functions are defined for DCL1, which is involved in miRNA biogenesis (Finnegan, 2003); DCL3 is required for retro-element and transposon siRNA production and chromatin silencing, and DCL2 has been implicated in viral siRNA production (Xie, 2004). The function of DCL4 is not known.

Quelling

This term is specific for transgene-induced PTGS in Neurospora crassa. The insert shows C.elegans embryos produced by worms that had been fed BIR-1 or dsRNA. BIR-1 deficiency produces a profound cytokinesis.

Endogenous mRNAs by microRNAs

They are endogenous, non-coding RNAs (18-25 nucleotides) existing both in plants and in animals (Reinhart, 2002), (Carrington, 2000). These small-RNA fragments work as the specificity determinant by being incorporated into the RNA-induced silencing complex (RISC) endonucleases (Hammond, 2000), which degrades mRNAs in a sequence-specific manner or inhibits protein translation (Tang, 2003).

Heterochromatic Silencing and non coding RNA

Heterochromatin has been ascribed important functions in gene regulation and chromosome architecture since the early days of cytogenetics. Heterochromatin decorates centromeres, telomeres, and nuclear organizers and can silence nearby genes in an unstable fashion leading to position effect variegation (PEV) (Lippman, Martienssen, 2004). At the sequence level, heterochromatin is composed of highly repetitive DNA with little or no coding potential, which sets it apart from euchromatin, its generich cousin. These contrasts parallel differences in transcriptional activity: euchromatin is actively transcribed, whereas heterochromatin was thought to be largely silent.

Biochemically, heterochromatic DNA is often methylated, while histones are hypoacetylated and methylated on residues associated with transcriptional repression (e.g., H3K9me). These epigenetic marks determine its tight packaging and recruit heterochromatin protein 1 (HP1), a suppressor of PEV. Centromeric heterochromatin is present in organisms as distantly related as fission yeast, plants, insects, and mammals. (Dawe, 2003). A notable exception is the budding yeast, whose centromeres are much reduced. Coincidentally, yeast has lost histone H3K9 methyltransferases and HP1, as well as all trace of the RNAi machinery.

RNA-Mediated Silencing of Transposable Elements

Transposable, or ''controlling'' elements was first discovered in maize more than half a century ago (McClintock, 1951). Since then TEs have been found in every eukaryotic genome, and in many cases constitute a majority of the sequence content. Active transposition is potentially mutagenic, and transpositions occurring in the germline are transmitted to the offspring, with cumulative defects in subsequent inbred generations.

Additionally, the insertion of a TE can affect the proper expression and processing of nearby genes (Lippman, 2004). The presence of TEs thus influences development, for example at the agouti locus in mice (Morgan, 1999). At the macroscopic level, polymorphic TE distribution and silencing could account for an unknown amount of intraspecies variability. In its most extreme form, unleashed transposition in the germline could be responsible for speciation itself. This was first proposed for hybrid dysgenesis in Drosophila and has recently been demonstrated in sunflower (Ungerer, 2006). TEs, and their epigenetic baggage, are therefore major players in evolution, and it is no surprise that genomes have acquired mechanisms to regulate TEs, especially in the germline. As a consequence, the vast majority of TEs are silent and inactive. This silencing is brought about by both transcriptional and posttranscriptional mechanisms.

Transposons were first discovered in plants, and TE regulation is well understood in plant models such as Arabidopsis thaliana. Plant TEs are methylated and associated with H3K9me2 and small RNA, indicating RNAi, DNA, and histone modification pathways are responsible for TE silencing. Twenty-four nucleotide siRNAs are derived from most TEs by DCL3 and depend on RdRP action by RDR2. However, most TEs are not upregulated in dcl3 and rdr2 mutants, while many are reactivated (and siRNA is lost) in mutants of the CpG DNMT met1 or the Swi2/Snf2 ATPase ddm1 (Lippman, 2003).

Transcriptional silencing downstream of DNA methylation requires the HDAC hda6/sil1 and a distantly related Swi2/ Snf2 ATPase, MOM1 (Mittelsten Scheid, 2002). When met1 and ddm1 are backcrossed to wild-type strains, most TEs remain active (resembling ''presetting'' of TEs in maize). The few TEs that are resilenced do not lose siRNA in the mutant, suggesting that siRNAs are needed in cis to initiate transcriptional silencing. Consistent with this model, hda6 mutants lose TE silencing but not siRNA, and silencing is readily reestablished after a backcross (Lippman, 2003).

RNA-Dependent DNA Methylation

DNA methylation in plants and animals requires both de novo methylation (of unmethylated DNA) and maintenance methylation (of hemimethylated DNA), and RNA has been implicated in guiding DNA methylation patterns. The activity of MET1 could maintain silencing even when the initial trans-acting trigger was removed (Matzke, Birchler, 2005), while the maintenance activity of CMT3 and SUVH4 was less stable, resulting in variegated plants in the absence of the inverted repeat (Ebbs, Bender, 2006; Melquist, Bender, 2004). Unlike symmetric CpG methylation, non-CpG methylation requires active signals to target regions of replicated DNA. DRM1 and DRM2 are redundant and were not recovered in forward screens, but reverse genetics showed that they are required for asymmetric CpNpN methylation and for

MicroRNAs (miRNAs)

It plays important roles in a broad range of biological processes including development, cellular differentiation, proliferation, and apoptosis. At least 100 miRNA genes have been identified in invertebrates, and 500-1000 in vertebrates and plants. Computational predictions of miRNA targets estimate that each miRNA regulates hundreds of different mRNAs, suggesting that a large proportion of the transcriptome is subject to miRNA regulation (Bushati, Cohen, 2007). To perform their regulatory functions; miRNAs assemble together with Argonaute family proteins into miRNA induced silencing complexes (miRISCs). Within these complexes, miRNAs guide Argonaute proteins to be fully or partially complementary mRNA targets, which are then silenced post transcriptionally (Bushati, Cohen, 2007).

Despite remarkable progress in our understanding of miRNA biogenesis and function, the mechanisms used by miRNAs to regulate gene expression remain under debate. Indeed, published studies indicate that miRNAs repress protein expression in four distinct ways: (1) co-translational protein degradation; (2) inhibition of translation elongation; (3) premature termination of translation (ribosome drop-off); and (4) inhibition of translation initiation.

In addition, animal miRNAs can induce significant degradation of mRNA targets despite imperfect mRNA-miRNA base-pairing. MicroRNAs might also silence their targets by sequestering mRNAs in discrete cytoplasmic foci known as mRNA processing bodies or P bodies, which exclude the translation machinery.

Cellular Proteins Involved in RNA Silencing in Plants

Several genes controlling RNA silencing in plants have been discovered through genetic screens of Arabidopsis mutants involved in transgene-induced RNA silencing. They encode an RNA-dependent RNA polymerase [SGS2/SDE1], which is a coiled-coil protein of undetermined function [SGS3] (P. Mourrain et al. 2000), a protein containing PAZ and Piwi domains [AGO1], besides an RNA helicase [SDE3]. Genes encoding related proteins are involved in RNA silencing in C. elegans and Neurospora or Chlamydomonas. Definitely, the putative RNA dependent RNA polymerase (SGS2/SDE1) is related to QDE-1 [Neurospora] and EGO-1 [C. elegans] (A. Smardon et al. 2000); the PAZ/Piwi protein AGO is related to QDE-2 [Neurospora] (C. Catalanotto et al. 2000) and RDE-1 [C. elegans]; and the RNA helicase SDE3 is related to SMG-2 [C. elegans] and MUT-6 [Chlamydomonas] (D. Wu-Scharf et al. 2000). It is assumed that the function of SGS3 is dedicated for plants since no related proteins were found to be encoded in the genome of C. elegans and Drosophila (since both undergo RNA silencing of their genes). Studies in C. elegans and in a Drosophila in vitro silencing system have identified two ribonucleases involved in RNA silencing in those organisms and suggest additional components of the RNA silencing pathway in plants. The MUT-7 gene of C. elegans (R. F. Ketting et al. 1999) encodes a protein similar to RNaseD (having 39359 exonuclease activity), whereas the Drosophila DICER gene (E. Bernstein et al. 2001) encodes a protein similar to RNase III (having dsRNA endonuclease activity). In the Drosophila in vitro RNA silencing system, the input dsRNA is cleaved by the RNase III-like enzyme (DICER) into 21 up to 25 nucleotide RNAs of both polarities (siRNAs). The siRNAs incorporate into a multicomponent silencing complex (RISC) in the Drosophila, where they act as guides to find the complementary RNAs. An Arabidopsis ortholog of the DICER gene (called either CAF, SIN1, or SUS1) has been identified (S. E. Jacobsen 1999). Unfortunately, a knockout in this gene is lethal to the embryo, indicating that it is absolutely required for plants. The presence of hypomorphic mutants like CAF or SIN1 being defective in RNA silencing, is not yet known. Studies in Arabidopsis and Neurospora indicate that changes at the DNA level is required for transgene-induced RNA silencing in plants and fungi. Using reverse genetics, Arabidopsis mutants called ddm1 and met1 were shown to be impaired in the triggering (ddm1) or maintenance (met1) of silencing of an exogenous 35S-GUS transgene (J. Morel, et al. 2000) The corresponding DDM1 and MET1 genes encode a SNF2/SWI2 chromatin remodeling factor and maintenance DNA methyltransferase, respectively (E. J. Finnegan et al. 1996). Although DNA methylation seems to be not necessary for quelling, because it occurs efficiently in the Neurospora dim-2 methylation mutant, a putative DNA helicase (QDE- 3) is required for quelling and could play a role similar to that played by DDM1 in Arabidopsis.

Post Transcriptional Gene Silencing In Plant

Post transcriptional gene silencing (PTGS) in plant is an RNA- degradation mechanism that shows similarities to RNA interference (RNAi) in animals. (Vaucheret et al. 2001) PTGS results in the specific degradation of a population of homologous RNAs. The basic requirements for RNA degradation in both animals and plants were found to have a great similarity as in the presence of double-stranded RNA (dsRNA), spreading within the organism from a localized area, the association with the accumulation of small interfering RNA (siRNA) and the presence of RNA-dependent RNA polymerases, RNA helicases besides, some proteins of undetermined functions, containing PAZ and Piwi domains. Concerning the mechanism of RNA-degradation in both animals and plants was also found to have common basics, so we could say the unifying feature of RNA silencing is, the cleavage of long double-stranded dsRNA into short-interfering (21-26 nt) RNAs by a Ribonuclease III-like enzyme termed DICER. In plants, four Dicer-like (DCL) enzymes have been identified in Arabidopsis thaliana; however, distinct functions are defined for DCL1, which is involved in miRNA biogenesis; DCL3 is required for retroelement and transposon siRNA production and chromatin silencing, and DCL2 has been implicated in viral siRNA production. The function of DCL4 is not known. MicroRNAs are endogenous, non-coding RNAs (18-25 nt) existing both in plants and in animals. These small-RNA fragments serve as the specificity determinant by being incorporated into the RNA-induced silencing complex (RISC) endonuclease, which degrades mRNAs in a sequence-specific manner or inhibits protein translation. However, some differences between both plants and animals are still quite clear. First, PTGS in plants requires at least two genes which are - SGS3 (which encodes a protein of unknown function containing a coil-coiled domain) and MET1 (which encodes a DNA methyltransferase) - that were found to be absent in C. elegans thus consequently they are not required for RNAi. Second, it was found that all Arabidopsis mutants that reveal impaired PTGS are hypersusceptible to infection by the cucumovirus CMV, indicating that PTGS participates in a mechanism for plant resistance to viruses. Although many viruses have developed strategies to counteract PTGS and could successfully infect the plants. On the cellular level, PTGS reduces mRNA accumulation in plant cytoplasm but does not affect transcription (de Carvalho et al., 1992; van Blockland et al., 1994). Detailed analyses of RNA content in plants carrying out PTGS has revealed the presence of several independent RNA degradation intermediates.

Initiation, Propagation and Maintenance

Studying how PTGS is started has showed the presence of basically three steps: initiation, propagation and maintenance (Vaucheret et al. 2001). Indeed, spontaneous initiation of PTGS by nitrate reductase, nitrite reductase or SAM-synthase (trans) genes (which leads to particular chlorotic or necrotic phenotypes that correlate with the disappearance of the corresponding RNA) starts with interveinal or vein-localized spots on one leaf and then propagates to the upper leaves, in which it is subsequently maintained (Boerjan et al., 1994; Palauqui et al., 1996). These non clonal patterns were found reproducibly in all transgenic lines in which a given gene was silenced, suggesting that the analysis of spontaneous PTGS into localized initiation, systemic propagation and active maintenance is a general rule.

Initiation

Spontaneous initiation of PTGS in transgenic plants is localized, so it is considered to be particularly difficult to study. Most data concerning the controlling the initiation are indirect and result from the analysis of parameters that increase or decrease the efficiency of spontaneous triggering of PTGS. Such studies have revealed that two types of transgene loci efficiently trigger PTGS. The first type corresponds to highly transcribed single transgene copies. It is suggested that the efficiency of triggering could depend on the probability that the transgene produces a particular form of RNA above a threshold level. Actually, PTGS is triggered mostly when plants are homozygous for the transgene locus (de Carvalho et al., 1992). In addition, PTGS is triggered more efficiently when strong promoters are used (Que et al., 1997). Finally, PTGS is inhibited when transgene transcription is blocked (Vaucheret et al., 1997). The second type of transgene loci that efficiently triggers PTGS is those carrying two transgene copies arranged as an inverted repeat (IR). These IRs are usually transcribed at very low levels, which argues against the threshold model (van Blockland et al., 1994). To explain their ability to efficiently trigger PTGS, investigators have proposed that these IRs produce dsRNA by read-through transcription and that dsRNA efficiently trigger PTGS, even when produced at a low level. Indeed, introduction of single transgene copies that have a panhandle structure (i.e. carry the same sequence cloned in sense and antisense orientations downstream of the promoter) leads to efficient silencing of homologous (trans)genes, which so such dsRNAs are efficient initiators of PTGS (Hamilton et al., 1998; Waterhouse et al., 1998). The above results coincided with the discovery of RNAi in animals, a process that results in specific RNA degradation induced by injection of homologous dsRNA (Fire et al., 1998) or expression of panhandle transgenes (Tavernarakis et al., 2000). These similarities suggest that PTGS in plants and RNAi in animals could derive from an ancestral mechanism allowing degradation of RNAs that are homologous to dsRNAs abnormally present in a cell. However, the PTGS mechanisms triggered by highly transcribed single transgene loci and transgene IRs in plants are (at least in part) different. Indeed, mutants that PTGS triggered by highly transcribed single transgene copies is impaired exhibit efficient PTGS triggered by transgene IRs (H.V. and P. Waterhouse, unpublished). This suggests that highly transcribed single transgene loci do not directly produce dsRNA and that the mutants that have been isolated are impaired in the steps leading to the formation of dsRNA.

Systemic propagation

The transmission of PTGS of nitrate reductase, nitrite reductase or SAM-synthase (trans) genes from localized interveinal spots or vein-localized to the upper leaves of plants suggested that a PTGS propagation signal exists. The existence of such a signal was clearly established by grafting. Silencing was transmitted with 100% efficiency from silenced stocks to target scions expressing the corresponding transgene but not to scions expressing a non-homologous transgene, which indicates that the signal is sequence-specific (Palauqui et al., 1997). Silencing of nitrate-reductase genes was also transmitted to a non-transgenic mutant scion over expressing the endogenous Nia2 gene owing to metabolic derepression but not to a wild-type scion, which indicates that over accumulation of Nia mRNA above the level of that in wild-type plants, rather than the presence of a transgene in the scion, is required for triggering of RNA degradation during PTGS (Palauqui and Vaucheret, 1998). The transmission of PTGS also occurred when silenced stocks and non-silenced target scions were physically separated by up to 30 cm of stem of a non-target wild-type plant, indicating long-distance propagation (Palauqui et al., 1997). Other scientists were able to draw a similar conclusions when PTGS of a GFP transgene was systemically triggered after they inoculated one leaf of a non-silenced GFP transgenic N. benthamiana plant with an Agrobacterium strain carrying the GFP transgene (Voinnet and Baulcombe, 1997) or biolistically introduced the GFP transgene (Voinnet et al., 1998). Because it is sequence specific and mobile, this signal could be made (at least in part) of RNA. Whether it corresponds to dsRNA or siRNA remains to be determined.

Maintenance

Grafting experiments using nitrate-reductase-silenced tobacco stocks and a set of different transgenic and non-transgenic scions revealed similar requirements for spontaneous initiation and maintenance. Indeed, when grafting-induced silenced scions were removed from the silenced stocks and regrafted onto wild type plants, silencing was not maintained in lines that cannot trigger PTGS spontaneously (Fig. 1, class I and III plants). These lines seem to be able to 'sense' the systemic PTGS signal that induces the degradation of the mRNA, but cannot produce the signal. On the other hand, silencing was maintained in transgenic lines that are able to trigger PTGS spontaneously (Fig. 1, class II plants), which indicates that only the transgene loci that are able to initiate PTGS can maintain a silent state.

The ability of a transgenic line to produce the systemic silencing signal could depend on the genomic location and/or the structure of the transgene locus, which would thus involve a nuclear step in PTGS. Chemical modifications (e.g. DNA methylation or histone acetylation) or structural modification (i.e. chromatin remodeling) could correspond to an epigenetic imprint induced by the systemic silencing signal, allowing PTGS to be actively maintained during development. This could be maintained in newly developing tissues in a conservative manner during replication or could be imposed de novo in each new cell in response to the systemic signal. Recently, it was showed that a mutant in which the major maintenance DNA-methyltranferase was impaired exhibited impaired maintenance of PTGS (Morel et al., 2000), which supports this hypothesis. The experiments described above clearly show that PTGS is a dynamic process that can be separated into initiation, propagation and maintenance. However, a number of points remain mysterious. In particular, the nature of the systemic silencing signal remains to be determined.

Fig. 1. Evidence for a systemic silencing signal and for a maintenance step in PTGS.

(A) Wild-type plants, (wt) do not undergo PTGS after grafting onto silenced transgenic plants. (B) Transgenic plants that do not spontaneously undergo PTGS (class I) undergo PTGS after grafting onto silenced transgenic plants but do not maintain silencing after elimination of the silenced rootstock. (C) Nonsilenced transgenic plants derived from lines that can spontaneously undergo PTGS (class II) undergo PTGS after grafting onto silenced transgenic plants and maintain silencing after elimination of the silenced rootstock. (D) Nontransgenic plants that express an endogenous gene at high level owing to metabolic derepression (class III) undergo PTGS after grafting onto silenced transgenic plants but do not maintain silencing after elimination of the silenced rootstock.

Geminiviruses and gene silencing in plants

Geminiviruses are characterized by having a small geminate particles (18 x 20 nm) containing either one or two circular ssDNA of about 2.7 kb Based on genome organization, host-range and vector specificity, the members of the family Geminiviridae are classified into four genera: Begomovirus, Mastrevirus, Curtovirus and Topocuvirus. For the Begomoviruses, they have 2 major components referred to DNA-A and DNA -B which are highly essential for their infectivity (Vanitharani et al 2005). For the DNA-A it has 6 genes named from AC1 to AC6, each encoding for a specific protein, but what we will discuss later is the activity of AC2 and AC4 so let us start by introducing their encoded products where AC2 encodes for a transcription activator protein (TrpAP) while for AC4 it encodes for a protein that has no function attributed to virus multiplication. Several plant viruses as Indian cassava mosaic virus (ICMV) and African cassava mosaic virus from Cameroon (ACMV-[CM]) were shown to initiate the PTGS system in infected plants with the production of virus-specific siRNAs. It is confusing how these viruses that do not have a dsRNA phase in their replication cycle can trigger PTGS in plants. The possibility that the bi-directional transcription of these viruses with transcripts occurring from opposite polarity overlap at their 30-ends was confirmed using strand-specific probes; the dsRNA formed in this way partly explains how these viruses induce PTGS in infected plants. Yet another possibility is that the strong fold-back structure of geminivirus transcripts could simply become a template for DICERs to cleave at specific locations and produce siRNAs. In plants, some virus-host interactions naturally lead to host recovery. The natural recovery responses induced by a nepovirus and a caulimovirus are similar to RNA-mediated virus resistance. This symptom recovery phenomenon is unusual for geminiviruses. However, in ACMV-[CM]-infected N. benthamiana and cassava, symptom recovery has been observed; in both cases mosaic formation and leaf distortion in the systemically infected leaves has been observed for 2-3 weeks after inoculation, after which the plants recovered from the infection symptoms. This recovery phenomenon is associated with the production of virus derived siRNAs beginning one week post-inoculation and becoming abundant in the newly developed symptom-less recovered leaves, both in N. benthamiana and cassava. This increase in virus-derived siRNA accumulation was accompanied by a reduction in the levels of both viral DNA and mRNA accumulation.

Geminiviruses can suppress the induced RNA silencing

Recently, 29 RNA silencing-inhibiting proteins that counter the antiviral RNA silencing have been identified in several plant viruses. Those proteins do not share homology but all might target similar or different step of RNA silencing pathway initiation, maintenance and systematic silencing that were previously explained. In 2005 Vanitharani et al. carried out Agrobacterium infiltration assay through which they were able to show that viruses being either recovery or non recovery type have different effect on the suppression of the induced PTGS. Concerning the recovery type viruses, showed a strong suppression activity unlike the non recovery type that were identified as induced RNA silencing suppressors.

A Novel Vector for Efficient Gene Silencing in Plants

The discovery that double-stranded RNA is responsible for posttranscriptional gene silencing (PTGS) in plants has guided Schattat et al. in 2004 to the development of a new generation of vectors for this purpose. There vector, named pJM007, allows a single pair of primers to be used in the directional cloning of sense and antisense cDNA strands. A crucial advance toward the development of genetic tools allowing optimal induction of PTGS has been the discovery that a linker fragment greatly increases the rate of observed silencing, particularly if a spliceable intron is situated between an inverted repeat consisting of the antisense and sense transcripts (Chuang and Meyerowitz, 2000; Smith et al. 2000). Further advances in this direction include the development of intron-containing vectors allowing a directional cloning of the sense and antisense transcripts (Phannibal, Wesley et al., 2001); vectors optimized for high-throughput gene silencing based on the Gateway recombinase technology and, most recently, vectors based on the generation of an inverted repeat of the 3′-untranslated region (Brummell et al., 2003). The pJM007 vector designed by Schattat et al. for PTGS induction is an intron-containing vector that revealed the ability to induce 95% PTGS in plants, ectopically expressing enhanced green fluorescent protein (EGFP). One of the unique features of pJM007 is the possibility to directionally clone the sense and antisense transcripts, taking advantage of 4 unique cloning sites limiting the intron sequence, 2 of them being rare 8-bp cutters.

The components of the new vector, including the backbone and the spliceable intron loop, were chosen from available sequences proven in practice to have a high degree of efficiency. As backbone for the vector the team has selected pRT-Ω/Not/Asc (Überlacker and Werr, 1996). This derivative of pRT100 (Töpfer et al., 1987) has several positive features for the required purpose. It comprises the 35S promoter sequence of cauliflower mosaic virus (CaMV) followed by the 67-bp Ω leader sequence of tobacco mosaic virus (TMV); 3 unique cloning sites consisting of NotI, BamHI, and XbaI; and the polyadenylation sequence of CaMV. Furthermore, the HindIII sites originally flanking the 35S promoter and the polyadenylation signal in pRT100 were both replaced by unique AscI sites (an 8-bp cutter), thus facilitating excision of the expression cassette and subsequent transfer into tDNA-based binary vectors such as those of the pGreen family. For the spliceable intron loop, a version of the second intron (IV2) of the ST-LS1 gene from potato was choosen, which was modified in its internal border sequences to better correspond to the consensus sequence for plant introns (Vancanneyt et al., 1990). The high splicing efficiency of this intron has been demonstrated, among others in an intron-containing β-glucuronidase gene (Vancanneyt et al., 1990) or in plant expression vectors (Ferrando et al., 2000). For the required purposes, the IV2 intron of the ST-LS1 gene has been amplified by PCR and introduced between the BamHI and XbaI cloning sites of pRT-Ω/Not/Asc. To allow the unidirectional cloning of the cDNA strand at the right side of the intron loop, a new FseI restriction site (a further unique 8-bp cutter) has been introduced between its border and the XbaI site (Figure 1a-b). The final vector, named pJM007, thus comprises 2 unique cloning sides at each side of the ST-LS1 intron. As represented in Figure 1c, the distribution of these 4 cloning sites permits the PCR amplification of the desired DNA sequence with a single pair of primers. Upon amplification, restriction with BamHI/NotI or FseI/XbaI directly allows the directional cloning of the amplified fragment both in the antisense and sense orientation.

Fig. 2. Map of pJM007.

Unique restriction sites are indicated. Those useful for the cloning of the sense and antisense transcripts at both sides of the intron loop are given in a larger font. The recognition sequences for 8-bp cutters are underlined. (b) Splice junction of the IV2 intron of the STLS1 gene in pJM007. The vector borders are capitalized, and the restriction sites are underlined. (c) Cloning strategy for a target gene using a unique pair of primers. These comprise the 4 recognition sequences for the 4 unique restriction enzymes present at the intron borders.

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