Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

Chaobo Tong,Shunping He,Baocheng Guo

doi:10.1186/1471-2164-10-83

Chaobo Tong, Shunping He + Show 1 more

Open Access

https://doi.org/10.1186/1471-2164-10-83

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Abstract

BackgroundShort and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening.ResultsA new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 × 105 and 1.7 × 105 per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts.ConclusionThe presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened the hypotheses containing the slippage model for initiation of reverse transcription, retropositional parasitism of SINEs on LINEs, the formation of the stem loop structure in 3'tail region of some SINEs and LINEs and the mechanism of template switching in generating new SINE family.

Highlights

Short and long interspersed elements (SINEs and long interspersed repetitive elements (LINE), respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology
The similar but not identical AB-polymerase chain reaction (PCR) sequences and intact A box and B box, together with its reasonable sequence similarity to certain tRNAs indicated that they may have been amplified from different short interspersed repetitive elements (SINE) copies of one same SINE family in the above two species
To amplify the regions flanking AB-PCR fragment and test whether the above AB-PCR fragments belong to a part of a certain SINEs as we expected, a pair of primer: primer IvF and IvR which face in opposite orientations and correspond to consensus sequences of AB-PCR was designed to perform inverse PCR in silver carp (Figure 1A, Additional file 1)

Summary

Introduction

Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Alu and LINE1 constitute more than 13% and 20% of human genome, respectively [1]. They proliferate and replicate themselves through a "copy and paste" mechanism called retroposition involving transcription of their genomic copies followed by reverse transcription of an RNA intermediate and resulting cDNAs reintegration at a new location into the genome host [2,3,4,5]. Over 100 LINE and nearly 100 SINE families have been described to date in various eukaryotic genomes [7]. So far SINEs appear to have gained novel functions, acting for example as enhancers or silencers that regulate the expression of preexisting functional genes [16,17,18]

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