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Retrotransposons are an evolutionarily ancient class of mobile genetic elements that transpose replicatively within their host genomes via RNA intermediates. The mobile element is transcribed by an RNA polymerase and then converted back into dsDNA by a reverse transcriptase (Lodish et al, 2005). Retrotransposons have been established to be ubiquitous and active components of the plant genome and are associated with numerous functions in the genome
Retroelements are active in a wide variety of genome functions from transcription to translation, DNA replication and intragene movements resulting in epigenetic modification (Shapiro et al, 2005). Insertion of these retroelements takes place closer to genes and they can alter the transcription and translation of the genes by their own regulators or promoters (Casacuberta et al, 2003). Not all element insertions are advantageous and beneficial, generating new regulatory elements could cause gene mutations that result in various diseases.
Retrotransposons are the major class of transposons in plants. In the last decade, a large scale effort was underway to sequence the genome of a model plant, Arabidopsis thaliana. This plant was chosen for the ambitious effort because of the exceptionally small size of its genome (approx. 130Mb).However, plants like Fritillaria (a member of the flowering plant family, Lilliaceae) which has the largest genome size in the plant kingdom (124,000 Mb) has received much less attention. In many plants, retrotransposons and rearranged retrotransposons comprise large proportions of their genomes (Heslop-Harrison et al 1997). In the large maize genome, highly and middle-repetitive sequences, together with low-copy-number retrotransposons, were estimated to comprise approximately 80% of the genome (Messing et al., 2004; SanMiguel et al., 1998). Plants with smaller genomes tend to accumulate fewer retrotransposon repeats and in the small and fully sequenced Arabidopsis genome, retrotransposons make up less than 10% of the genome (Arabidopsis Genome Initiative, 2000). This suggests that retroelements, particularly retrotransposons account for most of the great variation in plant genome size (San Miguel et al; 1996).
BACKGROUND OF PLANT ACTIVE ELEMENTS AND THEIR IMPORTANCE:
In plants, retroelements contribute to variation in genome size, which means genome size is dependent on the amount of repeated sequences (Kumekawa et al, 1999)
As seen in two Oryza sativa sps, the differential abundance of RIRE-I element in one of the species can account for nearly 1/3 rd of difference in the genome size (Casacuberta et al, 2003). This is due to the presence of active retroelements which replicate and can increase the copy number, thereby responsible to plant genome variation. Studies have shown that vertical and horizontal transmission of retroelements takes place in higher plants which plays a role in evolution of these mobile elements (Flavell et al, 1992). It is also possible that retroelements accumulate by means of unequal crossing over, replication slippage or preferential insertion into linked sites (Heslop- Harrison et al, 1997).
In majority of plants, retroelements are transcriptionally and transpositionally inactive and are fixed in genome locations. Active elements comprise only a minute fraction of the transposable element complementing the genomes of maize and of most other multicellular organisms. Active retroelements are transcribed actively and are highly polymorphic. In plants active elements have been found in tobacco (TNT1) (Grandbastien et al, 2005) and barley (BARE1) (Vincent et al, 2005). Retrotransposons that are largely inactive during development can be transcriptionally activated and transpose under conditions of biotic and abiotic stress. It is widely seen in plants that active retroelements are induced during stressfull conditions and change in microclimate.
In plants the type of retroelements examined are the Ty1-copia group and the gypsy group which is a class of retroelements that are flanked by long terminal repeats. These are known as (LTR) retrotransposons and the non-LTR retrotransposons consists of LINE elements. The life cycle of the LTR retrotransposons resembles that of retroviruses, i.e. it comprises transcription, translation, reverse transcription and integration of cDNA copy, back into the genome. LTRs encode proteins having RNA binding , endonuclease, and reverse transcriptase function (Turner and Summers, 1999). The LTRs contain the promoter necessary for transcription and specify the terminator and polyadenylation signals needed for RNA processing. Copia and Gypsy elements differ in the order of intergrase and reverse transcriptase domain arrangement in the protein coding region.
The Non-LTR lacks the integrase gene coding region; hence the protein coding region consists of gag and pol genes for enzymatic activities. They contain a promoter that is transcribed from an internal promoter (Casacuberta et al, 2003). Replication takes place at the integration site itself, where the endonuclease nicks the host DNA to generate a free 3' hydroxyl that serves as the primer for reverse transcription of the LINE (Boeke, 1997; Lenoir et al., 2001; Szafranski et al., 2004).
The Ty-1 copia elements are among the earliest retroelements found well studied in several plant species. These groups of elements are the most diverged of the LTR retroelments and studies have shown that these elements are highly heterogeneous and their extent of heterogeneity varies among plant species (Flavell et al, 1992).
Gypsy elements are structurally similar to retroviruses but are not as ubiquitous as copia elements. They are abundantly present and studies have indicated that have acquired the env gene enabling the evolution of retroviruses (Flavell et al, 1997).
LINE elements are found in the nuclear genomes of many plant species. However the LINE elements are not as much abundant in plants as seen in mammalian genomes. Studies indicate that probably majority of these elements are inactive or under the control of the host genome (Schmidt et al, 1999) .
To study the retrotrasposons Scoliopus bigelovii, a member of Liliceae family was done. Core Liliales include several beautiful and species-rich genera of bulbous monocots and is a good model to study genome expansion because of their large and varied genome size. Patterson & Givnish (2002) developed a robust phylogeny for core Liliales based on sequence data form the chloroplast-encoded and nucleus genes and formed a phylogenetic tree. The principal phylogenetic conclusion that emerged from the tree was that Prosartes is a closest relative to Scoliopus. However these species vary a lot in their genome sizes. Scoliopus genome is one of the largest~ >15GB where as Prosartes is <5 GB. It is an interesting research topic as to identify what is the factor that is responsible for this huge genome size variation. Thus it is a challenging attribute to research on Scoliopus species and to identify the transposable elements present in them and to compare them with experimental counterparts Prosartes, in order to study the mechanism played by the transposable elements, which is responsible for the variation in the genome size.
To identify the types of retroelements present in Scoliopus bigelovii sps first the composition of retroelements was done to determine the presence of copia, gypsy and line retroelements . Secondly, the obtained retroelements were sequenced and characterized quantitatively by Real time PCR. Phylogenetic analysis was done to investigate the evolutionary relationship between different sequences of a particular retroelement. Further the presence of active retrotransposons was identified in different parts of the plant (leaf, Flower) using PGI gene as a positive control, to determine the elements that can have influence on the evolution of genome not only by increasing genome size but also serving as substrates for rearrangements.
The copia , gypsy and the LINE retroelements was obtained from Scoliopus bigelovii leaf and flowers samples using degenerate primers and sequences was obtained.
Clone 1: >gi|439436|gb|M94479.1|LIRCOPIAC Liriodendron tulipifera copia-like retrotransposon reverse transcriptase gene, partial cds
Sequencher was used to edit sequences and tBLASTX and CLUSTALW was used and phylogenetic tree was developed to identify evolutionary relationship. The presence of active retrotransposons using degenerate primers was obtained.
Research is yet to be done using retroelement specific primers and their influence in genome expansion. This could be achieved by developing interspecies specific primers designed from the above obtained clone. These findings could help in understanding the retrotransposon responsible for naturally contributing to the interspecies gene flow, giving valuable data about genome evolution and phylogenetic relationships. This work also enhances our knowledge in understanding the role of retrotransposon in different plant species and organisms (humans). As the human genome consists of a higher percentage of these retroelements, an increased understanding of their properties could provide an insight in their evolution and the causes of genetic diseases.
Kumekawa, Norikazu, Eiichi, Ohtsubo, and Hisako(1999): Identification and Phylogenetic analysis of gypsy type retrotransposons in the plant kingdom. Genes Genetics System 74:299-307.
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