Estructuras subyacentes del diseño de páginas web

damages by ROS or nick generation, UV light specifically induces base dimers. The lesions include mismatched bases, double-strand breaks and chemically-modified bases (Britt,1996).

1.6.2 Repair mechanisms

There are different mechanisms of DNA repair, including the direct reversal of damage, the repair mechanism by excision and the repair mechanisms of double-strand breaks (Bray and West,2005).

Cyclobutane pyrimidine dimer (CPD) photolyases can directly reverse the main damage caused by UV-light exposure in a process called photo-reactivation. In rice, it has been shown that photo-reactivation is the preferential repairing mechanism in non-dividing tissues (Kimura et al.,2004). Furthermore,Kaiser et al.(2009) demonstrated that the over-expression of the CPD photolyase At-PHR1 (Ahmad et al.,1997) increased plant fitness under elevated UV-light radiation.

Repair mechanisms undergoing base or nucleotide excision are repaired using the information of the complementary strand. The existence of this repair mechanism in plants has been tested in cell extracts, suggesting that gap filling and ligation may proceed either through insertion of just one nucleotide or several nucleotides (Córdoba-Cañero et al.,2009). The complex CUL4-DDB1A, through its E3 ubiquitylation activity (Hu et al., 2004), is involved in the nucleotide excision repair (NER) mechanism. The deletion of DDB1 in mice has effects in cell cycle, cell death and embryonic development (Cang et al.,2006). The complex CUL4-DDB1A in Arabidopsis (Bernhardt et al.,2006) has also been shown to be involved in this repairing mechanism (Molinier et al.,2008).

Furthermore, WDR proteins interact with this complex, and specially PRL1 seems to be one of this interacting partners in Arabidopsis (Lee et al.,2008).

Double-strand break (DSB) repair uses information from identical or very similar DNA se-quences. The repair mechanisms can make use of homologous recombination and non-homologous end joining (Bleuyard et al.,2006). A single non-repaired DSB in a dispensable artificial chromo-some can produce cell death in yeast (Bennett et al.,1996). PSO4 (Prp19) was already associated with this repair mechanism (Henriques et al.,1989). Furthermore, hPRP19, together with hCDC5L, hPLRG1 and hSPF27 have been found to be related to the event of double-strand breaks caused by DNA inter-strand cross-links (Zhang et al.,2005). However, the ubiquitylated state of hPRP19 is critical: when ubiquitylated, hPRP19 has been shown to fail in interacting with either hCDC5L or hPLRG1 indicating that DNA damage can induce changes in the hPRP19 core complex (Lu and Legerski,2007).

1.7 Transposable elements in plants

Transposable elements (TE) were first discovered in plants. In the work published byMcClintock (1950) in maize, they were named as controlling elements, elements that could control the action of neighbouring genes. One of this elements, termed Dissociator, was found to cause the break of the chromosome upon dissociation. In general, these elements were found to move from one position to another in the linkage unit. Terms like variegation, mosaicism, mutable loci or positional effect could be explained by studying the function of various transposable elements.

1.7.1 Classification

TEs are classified into two categories which differ in the transposition process. Class Ⅰ are RNA transposons or retro-transposons, whereas class Ⅱ are DNA transposons (Feschotte et al.,2002).

Class Ⅰ transposons (retro-transposons) replicate in the host genome by a copy and paste mechanism. The original element is not excised from the host genome and its mRNA is trans-formed into a new element upon integration elsewhere. Two groups of retro-transposons are distinguished by the presence or absence of flanking long terminal repeats (LTRs). To the LTR sub-group belong the Ty1/copia and the Ty3/gypsy families which have a similar organization to retro-viruses. The second sub-group does not exhibit LTRs but a polyadenylation site at the 3’ end, the LINE (long interspersed nuclear elements) and SINE (short interspersed nuclear elements) families belong to this group (Figure1.4,Wicker et al.,2007).

Class I(retro-transposons) Ty1/copia

Ty3/gypsy Retrovirus

Class II(DNA transposons)

Mariner Helitron Maverick

Figure 1.4: Classification of transposable elements families. : long terminal repeat; : coding region; : terminal inverted repeats; : non-coding region; : region containing aditional ORF(s); : diagnostic feature in non-coding region.

GAG: capsid protein; AP: aspartic proteinase; INT: integrase; RT:

reverse transcriptase; RH: RNase H; ENV: envelope protein; Tase*:

transposase; RPA: replication protein A; Y2 HEL: helicase; C-INT:

cysteine protease; POL B: DNA polymerase B. Adapted fromWicker et al.(2007).

Class Ⅱ transposons (DNA transposons) are not transcribed like class Ⅰ. Instead, they are excised and reinserted somewhere else in the genome. This class includes three groups (Feschotte and Pritham, 2007): 1. The classic DNA transposons which are transposed by cut and paste mechanism by an element-encoded transposase, are transposed as double strand DNA and have, generally, terminal inverted repeats. After transposition they leave a characteristic repeated sequence from the flanking host sequence. 2. The group of Helitrons, originally discovered in plants (Kapitonov and Jurka,2001), which have a different transposition system –rolling-circle–

that showed to have implications in capturing host gene fragments (Hollister and Gaut,2007). 3.

The group of Maverick-like transposons has no examples described in plants. Class Ⅱ transposons

1.7. TRANSPOSABLE ELEMENTS IN PLANTS 1. INTRODUCTION

can only increase their number during DNA replication. When the transposon is in one DNA area that has already been replicated and jumps to other area which has not undergo replication.

1.7.2 Life cycle of LTR retro-transposons

LTR retro-transposons are the main components of higher plant genomic DNA and by far, the best studied examples (Kumar and Bennetzen,1999). Retro-transposition involves four steps:

transcription, translation, reverse transcription of mRNA to cDNA and integration of the cDNA in the host genome.

First, the retro-transposon is transcribed into RNA by the host RNA polymerase Ⅱ using the promoter placed in the 5’ LTR. The poly-cistronic mRNA is translated into the proteins that from the virus-like particle, reverse-transcribe the RNA and integrate the cDNA. Two RNA molecules are usually packed into the virus-like particle and are transcribed subsequently into double strand cDNA (dsDNA). Priming for dsDNA synthesis is done by tRNA and the 5’ and 3’ LTR RNA sequences. Finally, the dsDNA is integrated into the host DNA, adding another copy of the transposable element to the genome (Havecker et al.,2004).

This process is tightly controlled, involving functions encoded by the element itself and by the host. One of the major control steps is transcription, which determines both the production of the template RNA required in the reverse transcription and the synthesis of mRNAs for translation. In LTR retro-transposons, transcriptional control involves cis-regulatory sequences that are normally found in the LTR region of the element (Casacuberta and Santiago,2003). Promoter sequences of the 5’ LTR region have been shown to be similar to plant defence promoters, moreover, proteins induced by pathogen-related stresses interact in vivo with this sequences (Vernhettes et al.,1997).

1.7.3 Retro-transposons in the context of a host genome

Many studied plant retro-transposons are normally silent during plant development but activated by stresses, like pathogen attack, energy-depriving conditions, wounding, or in vitro culture (Grandbastien,1998). McClintock already proposed that this process may reflect a plant strategy to increase the genomic diversity, being the retro-transposons the direct generators of this diversity (McClintock,1950).

AlreadyGrandbastien et al.(1997) observed the connection of stress responses in plants with the activation of retro-transposon transcription, especially stresses caused by pathogen attack.

It was already observed that regulatory cis-elements of several known retro-transposons share similarities with a motif involved in the activation of several plant defence genes (Vernhettes et al.,1997). More recently,Maumus et al.(2009) made the connection of stress conditions with genome re-arrangement due to LTR retro-transposon activity: they showed that in the diatom algae (Phaeodactylum tricornutum), LTR retro-transposons are very abundant and are activated in several stress conditions. Furthermore, different accessions from around the world show different insertion patterns, which suggest the generation of intra-specific genetic variability through genome re-arrangement.

Retro-transposons are widespread in plant genomes in high copy number, representing a 4–8 % of the genome of Arabidopsis to a 50–80 % in the case of maize (Kumar and Bennetzen, 1999). The example of the Ty1/copia Hunk-2 has an estimated copy number of 200 000 in maize

(Meyers et al., 2001). However, they discuss that elements with high-copy number are not expressed, whereas elements with low-copy number are usually transcriptionally active. A cause of –endogenous– activation of transposons is DNA methylation. Mutants of Arabidopsis deficient for DNA methylation were shown to have a higher number of integrated copies of one class of retro-transposons (Miura et al.,2001).