Elsevier

Gene

Volume 448, Issue 1, 1 December 2009, Pages 40-45
Gene

Multiple chicken repeat 1 lineages in the genomes of oestroid flies

https://doi.org/10.1016/j.gene.2009.08.010Get rights and content

Abstract

Retrotransposons including CR1 (chicken repeat 1) elements are important factors in genome evolution. They also mobilize in a genome in a way that makes them useful for phylogenetic analysis and species identification. This study was designed to identify lineages of CR1 elements in the genomes of forensically important oestroid flies and to further characterize one family, Sbul.CR1B. CR1 fragments from several taxa were amplified, cloned, sequenced and analyzed to identify different lineages of elements. A variety of retrotransposon families were recovered that exhibit similarity to known retrotransposon families. A number of these lineages may have given rise to taxon-specific subfamilies that have been recently active in oestroid fly genomes. One element from Sarcophaga bullata was analyzed in detail to reconstruct a partial Open Reading Frame containing both the reverse transcriptase (RT) and endonuclease (EN) domains. These domains were used to identify conserved amino acid regions in the recovered consensus via comparison to known non-LTR retrotransposons. Phylogenetic analysis of the RT domain revealed the recovered ORF in S. bullata compares favorably with previously documented CR1-like elements. This work will serve as the basis for additional analyses targeted at developing a simple, efficient marker system for the identification of forensically important carrion flies.

Introduction

The retrotransposons, Class I transposable elements (TEs), utilize a “copy and paste” method of movement involving an RNA intermediate (Deininger and Batzer, 2002). Briefly, this process involves transcription into messenger RNA (mRNA) by RNA polymerase II and then reverse transcription and reincorporation into the genome.

Retrotransposons can be further subdivided into LTR (long terminal repeat) and non-LTR retrotransposons. Within the non-LTR group, LINEs (long interspersed elements) are autonomous representatives in that they encode at least some of the enzymes necessary for their mobilization. Although their structure is variable, LINEs usually contain one or two open reading frames (ORFs). The function of ORF1 likely encodes a protein with RNA binding and chaperone activity (Hohjoh and Singer, 1996, Martin et al., 2005). ORF2 is well known to encode an enzyme essential for retrotransposition via a process termed target primed reverse transcription (TPRT) (Luan et al., 1993). Phylogenetic analysis of the RT portion of LINEs has enabled differentiation among classes of elements. Eleven clades of LINEs have been identified based on analysis of the entire RT domain: Jockey, chicken repeat 1 (CR1), LINE 1 (L1), CRE, I, R1, R2, R4, RTE, Tad and LOA (Malik et al., 1999).

CR1-like elements are a widely distributed family of LINEs that have been identified in a variety of organisms including vertebrates — e.g., birds, reptiles and fish (Kajikawa et al., 1997, Haas et al., 2001, Sirijovski et al., 2005, Wicker, 2005, Shedlock, 2006, Watanabe, 2006) as well as invertebrates. Invertebrates with CR1-like TEs include the roundworm Caenorhabditis elegans (Jurka et al., 2005), the sea urchin Strogylocentrotus purpuratus (Jurka et al., 2005), and the human blood fluke Schistosoma mansoni (Drew and Brindley, 1997). Arthropods known to harbor CR1-like elements include Order Scorpiones (scorpions) (Glushkov et al., 2006), and Maculinea (butterflies) (Novikova et al., 2007), as well as the dipteran family Drosophila (Biedler and Tu, 2003), among many others. Very little work has been published with regard to transposable elements in Oestroidea, a large superfamily within Diptera. What little exists comprises studies detailing two Class II elements in Lucilia cuprina — a hAT-like element (Coates et al., 1996) and a transposon resembling the P-element of Drosophila (Perkins and Howells, 1992). Until now, the retrotransposon landscape of carrion fly genomes (Diptera: Calliphoridae) has gone uninvestigated.

Retrotransposons in general (Murata et al., 1993, Nishihara et al., 2005, Ray, 2005, Kaiser et al., 2007) and CR1 elements in particular (Watanabe et al., 2006) have been shown to be very reliable for species identification and for resolving species phylogeny questions. Because of their ability to insert essentially anywhere in the genome, it is very unlikely that any two elements would insert randomly at the same site in two different genomes. This virtually eliminates the chance of identity-by-state homoplasy (Batzer and Deininger, 2002, Salem et al., 2005, Ray et al., 2006). Furthermore, simple PCR reactions with locus-specific primers can be used to produce species-specific banding patterns and thereby establish species identity in unknown samples (Herke et al., 2007).

Using such a simple system to identify species would be especially useful in dipteran insects where it is difficult for a non-expert to identify taxa and where large, complex taxonomic assemblages are common. The order Diptera is one of the largest insect orders and representatives can be found in almost every habitat (Byrd and Castner, 2001). A major superfamily within Diptera is the Oestroidea. Among the oestroid flies are two families that are extremely important in forensic investigations where PMI (post mortem interval) is estimated (Byrd and Castner, 2001). These are Calliphoridae (∼ 1000 species), which are collectively known as blow flies, and Sarcophagidae (∼ 2000 species), the flesh flies.

We investigated the presence of CR1 elements in genomes from these two families and found that CR1 elements are indeed present in the genomes of interest and that multiple lineages are often harbored. Furthermore, evidence suggests that the elements may have been active relatively recently and would therefore be useful in the development of taxon-specific insertion loci.

Section snippets

Sample collection and preparation

DNA was extracted from six carrion fly taxa representing the two target families — Calliphora vicina, Calliphora vomitoria, Phormia regina, Cochliomyia macellaria, and Lucilia sericata of family Calliphoridae, and Sarcophaga bullata of family Sarcophagidae. Adult specimens were collected from the wild in Morgantown, WV, using raw liver as bait and a standard sweep net. Species identifications were confirmed using a dichotomous key (Whitworth, 2006) and specimens were preserved in 95% ethanol

CR1 amplification

PCR analysis using the primers CR1-A and CR1-S produced a single discrete band for each taxon examined. Each amplicon was found to be ∼ 550 bases, which was the expected length. Some resulting sequences showed no similarity to at least two other sequences and were therefore excluded from further analysis. The number of analyzed sequences obtained from each taxon and the number of haplotypes observed are shown in Table 2. For most taxa (C. macellaria being the lone exception) at least two

Discussion

While in mammals, one typically finds only a single lineage of active non-LTR retrotransposons, having multiple lineages of active non-LTR elements is not uncommon in non-mammalian animal taxa (Furano et al., 2004). For example, 21 families of non-LTR retrotransposons were found to have been recently active in the genome of the African malaria mosquito, A. gambiae (Biedler and Tu, 2003). While not as extreme an example, most of the species examined here also appear to harbor multiple recently

Acknowledgements

J.D. Wells kindly provided tissue samples for this work. B. Singh, C. Picard, and A. Nichols provided expertise in fly collection, identification, and dissection. J. D. Wells, S. DiFazio, H. Pagan, J. Smith, and R. Platt provided valuable comments on earlier drafts. This work was supported by the Eberly College of Arts and Sciences at West Virginia University (DAR) and by a grant from the WVU Faculty Senate (DAR).

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    Present Address: Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, MS 39762, USA.

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