Chicken Repeat Research Articles

A Universal Method for the Study of CR1 Retroposons in Nonmodel Bird Genomes

Presence/absence patterns of retroposon insertions at orthologous genomic loci constitute straightforward markers for phylogenetic or population genetic studies. In birds, the convenient identification and utility of these markers has so far been mainly restricted to the lineages leading to model birds (i.e., chicken and zebra finch). We present an easy-to-use, rapid, and cost-effective method for the experimental isolation of chicken repeat 1 (CR1) insertions from virtually any bird genome and potentially nonavian genomes. The application of our method to the little grebe genome yielded insertions belonging to new CR1 subfamilies that are scattered all across the phylogenetic tree of avian CR1s. Furthermore, presence/absence analysis of these insertions provides the first retroposon evidence grouping flamingos + grebes as Mirandornithes and several markers for all subsequent branching events within grebes (Podicipediformes). Five markers appear to be species-specific insertions, including the hitherto first evidence in birds for biallelic CR1 insertions that could be useful in future population genetic studies.

Marek’s disease virus challenge induced immune-related gene expression and chicken repeat 1 (CR1) methylation alterations in chickens

Marek’s disease virus (MDV) challenge induces lymphoma in susceptible chickens. Host genes, especially immune related genes, are activated by the virus. DNA methylation is an epigenetic mechanism that governs gene transcription. In the present study, we found that expression of signal transducer and activator of transcription 1 (STAT1) was upregulated at 10 days post infection (dpi) in MD susceptible chickens, whereas interleukin 12A (IL12A) was elevated in both resistant and susceptible chickens. However, we did not observe MDV-induced DNA methylation variations at the promoter CpG islands (CGIs) in STAT1 and IL12A. Interestingly, the methylation levels at Chicken Repeat 1 (CR1), the transposable elements (TEs) located upstream of two genes, were different between resistant and susceptible chickens. Furthermore, a mutation was identified in the CR1 element near IL12A. The impact of the point mutation in transcriptional factor binding is to be examined in the near future.

Cloning and expression analysis of piRNA-like RNAs: adult testis-specific small RNAs in chicken

Many small RNAs have been cloned from animal gonads, for example, endogenous small interfering RNAs (endo-siRNAs) were found in oocytes and piwi-interacting RNAs (piRNAs) were found in testis. Gallus gallus (chicken) is an important model organism, but few small RNAs have been identified from its gonads. In this study, we isolated and cloned 156 small RNAs from adult chicken testes. Since there is a reasonably even distribution from 22 to 33 nt, these small RNAs are slightly longer than miRNAs and endo-siRNAs. Genome mapping indicated that these small RNAs were derived from intergenic regions, exons, introns, and repetitive elements including chicken repeat 1, long terminal repeats, and simple repeats. Since they are similar with piRNAs, we named them piRNA-like RNAs (pil-RNAs). Northern blotting of 16 selected sequences showed that nine are specifically expressed in the adult testis. The vast majority of these pil-RNAs are poorly conserved between species, suggesting that they are unique to the adult chicken testis. Further analysis of the cloned pil-RNAs will improve our understanding of the function of small RNAs in animal gonad development.

Insertion of an intact CR1 retrotransposon in a cadherin gene linked with Bt resistance in the pink bollworm, Pectinophora gossypiella

Three mutations in the Pectinophora gossypiella cadherin gene PgCad1 are linked with resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac. Here we show that the r3 mutation entails recent insertion into PgCad1 of an active chicken repeat (CR1) retrotransposon, designated CR1-1_Pg. Unlike most other CR1 elements, CR1-1_Pg is intact, transcribed by a flanking promoter, contains target site duplications and has a relatively low number of copies. Examination of transcripts from the PgCad1 locus revealed that CR1-1_Pg disrupts both the cadherin protein and a long noncoding RNA of unknown function. Together with previously reported data, these findings show that transposable elements disrupt eight of 12 cadherin alleles linked with resistance to Cry1Ac in three lepidopteran species, indicating that the cadherin locus is a common target for disruption by transposable elements.

Multiple chicken repeat 1 lineages in the genomes of oestroid flies

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.

Calibration of Mutation Rates Reveals Diverse Subfamily Structure of Galliform CR1 Repeats

Chicken Repeat 1 (CR1) repeats are the most abundant family of repeats in the chicken genome, with more than 200,000 copies accounting for ∼80% of the chicken interspersed repeats. CR1 repeats are believed to have arisen from the retrotransposition of a small number of master elements, which gave rise to the 22 CR1 subfamilies as previously reported in Repbase. We performed a global assessment of the divergence distributions, phylogenies, and consensus sequences of CR1 repeats in the chicken genome. We identified and validated 57 chicken CR1 subfamilies and further analyzed the correlation between these subfamilies and their regional GC contents. We also discovered one novel lineage-specific CR1 subfamilies in turkeys when compared with chickens. We built an evolutionary tree of these subfamilies and concluded that CR1 repeats may play an important role in reshaping the structure of bird genomes.

Identification of novel CR1 subfamilies in an avian order with recently active elements

Chicken repeat 1 (CR1) is a taxonomically widespread non-LTR retrotransposon. Recent CR1 retrotranspositions in waterfowl suggested that, unlike chicken at least one subfamily remains active. Based on sequence information from 143 CR1 loci, six distinct groups of CR1 within the waterfowl coscoroba each with unique 3′ untranslated regions and distinct open reading frames are described. Through comparison to other previously described avian CR1 subfamilies, it is shown that five of the six coscoroba groups represent new subfamilies. At least one of these subfamilies is likely active and provides a target for future isolation of the first active member of this taxonomically widespread non-LTR family.

Recent CR1 non-LTR retrotransposon activity in coscoroba reveals an insertion site preference

BackgroundChicken repeat 1 (CR1) is a taxonomically widespread non-LTR retrotransposon. Insertion site bias, or lack thereof, has not been demonstrated for CR1. Recent CR1 retrotranspositions were used to examine flanking regions for GC content and nucleotide bias at the insertion site.ResultsElucidation of the exact octomer repeat sequence (TTCTGTGA) allowed for the identification of younger insertion events. The number of octomer repeats associated with a CR1 element increases after insertion with CR1s having one octomer being youngest. These young CR1s are flanked by regions of low GC content (38%). Furthermore, a bias for specific bases within the first four positions at the site of insertion was revealed.ConclusionThis study focused on those loci where the insertion event has been most recent, as this would tend to minimize noise introduced by post-integration mutational events. Our data suggest that CR1 is not inserting into regions of higher GC content within the coscoroba genome; but rather, preferentially inserting into regions of lower GC content. Furthermore, there appears to be a base preference (TTCT) for the insertion site. The results of this study increase the current level of understanding regarding the elusive CR1 non-LTR retrotransposon.

Phylogenomic Investigation of CR1 LINE Diversity in Reptiles

It is unlikely that taxonomically diverse phylogenetic studies will be completed rapidly in the near future for nonmodel organisms on a whole-genome basis. However, one approach to advancing the field of "phylogenomics" is to estimate the structure of poorly known genomes by mining libraries of clones from suites of taxa, rather than from single species. The present analysis adopts this approach by taking advantage of megabase-scale end-sequence scanning of reptilian genomic clones to characterize diversity of CR1-like LINEs, the dominant family of transposable elements (TEs) in the sister group of mammals. As such, it helps close an important gap in the literature on the molecular systematics and evolution of retroelements in nonavian reptiles. Results from aligning more than 14 Mb of sequence from the American alligator (Alligator mississippiensis), painted turtle (Chrysemys picta), Bahamian green anole (Anolis smaragdinus), Tuatara (Sphenodon punctatus), Emu (Dromaius novaehollandiae), and Zebra Finch (Taeniopygia guttata) against a comprehensive library approximately 3000 TE-encoding peptides reflect an increasing abundance of LINE and non-long-terminal-repeat (non-LTR) retrotransposon repeat types with the age of common ancestry among exemplar reptilian clades. The hypothesis that repeat diversity is correlated with basal metabolic rate was tested using comparative methods and a significant nonlinear relationship was indicated. This analysis suggests that the age of divergence between an exemplary clade and its sister group as well as metabolic correlates should be considered in addition to genome size in explaining patterns of retroelement diversity. The first phylogenetic analysis of the largely unexplored chicken repeat 1 (CR1) 3' reverse transcriptase (RT) conserved domains 8 and 9 in nonavian reptiles reveals a pattern of multiple lineages with variable branch lengths, suggesting presence of both old and young elements and the existence of several distinct well-supported clades not apparent from previous characterization of CR1 subfamily structure in birds and the turtle. This mode of CR1 evolution contrasts with historical patterns of LINE 1 diversification in mammals and hints toward the existence of a rich but still largely unexplored diversity of nonavian retroelements of importance to advancing both comparative vertebrate genomics and amniote systematics.

Molecular evolution of toxin genes in Elapidae snakes

The venom of the sea krait, Laticauda semifasciata, consists primarily of two toxic proteins, phospholipase A(2) (PLA(2)) and a three-finger-structure toxin. We have cloned both toxic protein genes, including the upstream region. PLA(2) genes contain three types of inserted sequences: an AG-rich region, a chicken repeat 1-like long interspersed nucleotide element sequence and an intron II 3' side repeat sequence. The molecular divergence of L. semifasciata PLA(2) genes was defined on the basis of the inserted sequences and their sequence homology. The length of intron I in the three-finger-structure toxin genes differs from species to species. The alignment analysis of intron I of the three-finger-structure toxin genes revealed that the intron I sequence of the ancestral gene comprised ten genetic regions. A hypothetical evolutionary process for the three-finger-structure toxin genes has also been developed.

Specific chicken repeat 1 (CR1) retrotransposon insertion suggests phylogenetic affinity of rockfowls (genus Picathartes) to crows and ravens (Corvidae)

While the monophyly of the order Passeriformes as well as its suborders suboscines (Tyranni) and oscines (Passeri) is well established, both on morphological and molecular grounds, lower phylogenetic relationships have been a continuous matter of debate, especially within oscines. This is particularly true for the rockfowls (genus Picathartes), which phylogenetic classification has been an ongoing puzzle. Sequence-based molecular studies failed in deriving unambiguously resolved and supported hypotheses. We present here a novel approach: use of retrotransposon insertions as phylogenetic markers in passerine birds. Chicken repeat 1 (CR1) is the most important non-LTR retrotransposon in birds. We present two truncated CR1 loci in passerine birds, not only found in representatives of Corvinae (jays, crows and allies), but also in the West-African Picathartes species which provide new evidence for a closer relationship of these species to Corvidae than has previously been thought. Additionally, we show that not only the absence/presence pattern of a CR1 insertion, but also the CR1 sequences themselves contain phylogenetic information.

Tuatara (Sphenodon) Genomics: BAC Library Construction, Sequence Survey, and Application to the DMRT Gene Family

The tuatara (Sphenodon punctatus) is of "extraordinary biological interest" as the most distinctive surviving reptilian lineage (Rhyncocephalia) in the world. To provide a genomic resource for an understanding of genome evolution in reptiles, and as part of a larger project to produce genomic resources for various reptiles (evogen.jgi.doe.gov/second_levels/BACs/our_libraries.html), a large-insert bacterial artificial chromosome (BAC) library from a male tuatara was constructed. The library consists of 215 424 individual clones whose average insert size was empirically determined to be 145 kb, yielding a genomic coverage of approximately 6.3x. A BAC-end sequencing analysis of 121 420 bp of sequence revealed a genomic GC content of 46.8%, among the highest observed thus far for vertebrates, and identified several short interspersed repetitive elements (mammalian interspersed repeat-type repeats) and long interspersed repetitive elements, including chicken repeat 1 element. Finally, as a quality control measure the arrayed library was screened with probes corresponding to 2 conserved noncoding regions of the candidate sex-determining gene DMRT1 and the DM domain of the related DMRT2 gene. A deep coverage contig spanning nearly 300 kb was generated, supporting the deep coverage and utility of the library for exploring tuatara genomics.

Insertion Events of CR1 Retrotransposable Elements Elucidate the Phylogenetic Branching Order in Galliform Birds

Using standard phylogenetic methods, it can be hard to resolve the order in which speciation events took place when new lineages evolved in the distant past and within a short time frame. As an example, phylogenies of galliform birds (including well-known species such as chicken, turkey, and quail) usually show low bootstrap support values at short internal branches, reflecting the rapid diversification of these birds in the Eocene. However, given the key role of chicken and related poultry species in agricultural, evolutionary, general biological and disease studies, it is important to know their internal relationships. Recently, insertion patterns of transposable elements such as long and short interspersed nuclear element markers have proved powerful in revealing branching orders of difficult phylogenies. Here we decipher the order of speciation events in a group of 27 galliform species based on insertion events of chicken repeat 1 (CR1) transposable elements. Forty-four CR1 marker loci were identified from the draft sequence of the chicken genome, and from turkey BAC clone sequence, and the presence or absence of markers across species was investigated via electrophoretic size separation of amplification products and subsequent confirmation by DNA sequencing. Thirty markers proved possible to type with electrophoresis of which 20 were phylogenetically informative. The distribution of these repeat elements supported a single homoplasy-free cladogram, which confirmed that megapodes, cracids, New World quail, and guinea fowl form outgroups to Phasianidae and that quails, pheasants, and partridges are each polyphyletic groups. Importantly, we show that chicken is an outgroup to turkey and quail, an observation which does not have significant support from previous DNA sequence- and DNA-DNA hybridization-based trees and has important implications for evolutionary studies based on sequence or karyotype data from galliforms. We discuss the potential and limitations of using a genome-based retrotransposon approach in resolving problematic phylogenies among birds.

A CR1 element is embedded in a novel tandem repeat (HinfI repeat) within the chicken genome

Highly repetitive DNA sequences constitute a significant portion of most eukaryotic genomes, raising questions about their evolutionary origins and amplification dynamics. In this study, a novel chicken repetitive DNA family, the HinfI repeat, was characterized. The basic repeating unit of this family displays a uniform length of 770 bp, which was defined by the recognition site of HinfI. The HinfI repeat was specifically localized in the pericentric region of chromosome 4 by fluorescence in situ hybridization and constitutes 0.51% of the chicken genome. Interestingly, a chicken repeat 1 (CR1) element has been identified within this basic repeating unit. Like other CR1 elements, this CR1 element also displays typical retrotransposition characteristics, including a highly conserved 3' region and a badly truncated 5' end. This direct evidence from sequence analysis, together with our Southern blot results, suggests that the HinfI repeat may originate from a unique region containing a retrotransposed CR1 element.

Cytogenetic repartition of chicken CR1 sequences evidenced by PRINS in Galliformes and some other birds

Chicken repeat 1 (CR1) belongs to the non-long repeat class of retrotransposons. Nearly 100000 repeats interspersed in the chicken genome are subdivided into at least six distinct subfamilies, each 300 bp long and all sharing substantial sequence similarity. CR1-like elements were found in genomes from invertebrates to mammals, suggesting their importance for genome structure and/or function. Moreover, numerous data support the hypothesis of their implication in regulation of gene expression. So, the chromosomal distribution of these CR1 sequences in vertebrates is of great interest to improve our knowledge about the genome structure, function and evolution. A comparison of the cytogenetic distribution of CR1 sequences was performed by PRINS using consensus chicken primers on the chromosomes of chicken and species of several bird orders: Galliformes, Anseriformes, Passeriformes and Falconiformes. The study revealed that CR1 repeats are spread over nearly all chicken chromosomes with a higher density on the macrochromosomes and in particular with hot spots on subtelomeric regions of chromosome 1, 2, 3q, 4q, 5q. Their distribution on the macrochromosomes forms a kind of banding pattern, which was not systematically matched with R- or G-banding. This banding pattern appears to be conserved on the chromosomes of the Galliformes studied, irrespective of their karyotypes, rearranged or not. CR1 primers also show similar signals on the chromosomes of birds phylogenetically more distant (Anseriformes, Passeriformes and Falconiformes). This fact confirms the importance of these sequences at the large scale of bird evolution and in the chromosomal structure. The location of CR1 sequences, and in particular of the hot spots, mainly within the richest CG areas are in conformity with the data on an epigenetic role of these highly conserved sequences.

Molecular evidence for hybridization of species in the genusGallusexcept forGallus varius

A phylogenetic tree for fowl including chicken in the genus Gallus and based on mitochondrial D-loop analysis further supports the hypothesis developed from morphology and progeny production that red junglefowl (RJF) is the direct ancestor of the chicken. The phylogenetic positions of the chicken and the other fowl species in the genus Gallus are of great importance when considering maintenance and improvement of chicken breeds through introgression of genetic variation from wild-type genomes. However, because the phylogenetic analysis based on the DNA sequences is not sufficient to conclude the phylogenetic positions of the fowls in the genus, in the present study, we have determined sequences of whole mitochondrial DNA (mtDNA) and two segments of the nuclear genome (intron 9 of ornithine carbamoyltransferase, and four chicken repeat 1 elements) for the species in the genus Gallus. The phylogenetic analyses based on mtDNA sequences revealed that two grey junglefowls (GyJF) were clustered in a clade with RJFs and chicken, and that one GyJF was located in a remote position close to Ceylon junglefowl (CJF). The analyses based on the nuclear sequences revealed that alleles of GyJFs were alternatively clustered with those of CJF and with those of RJFs and chicken. Alternative clustering of RJF and chicken alleles were also observed. These findings taken together strongly indicate that inter-species hybridizations have occurred between GyJF and RJF/chicken and between GyJF and CJF.

A recent chicken repeat 1 retrotransposition confirms the Coscoroba–Cape Barren goose clade

Chicken repeat 1 (CR1) is a member of the non-long terminal repeat class of retrotransposons. We have isolated a truncated CR1 element within the third intron of the lactate dehydrogenase B gene of the coscoroba and the Cape Barren goose (Anseriformes; Coscoroba coscoroba, Cereopsis novaehollandiae). Because the element was absent in orthologous loci within mallard ( Anas platyrhynchos), snow goose ( Anser caerulescens), and tundra swan ( Cygnus columbianus), it provides strong support to the recent novel proposal by Donne-Goussé et al. [Donne-Goussé, C., Laudet, V., Hänni, C., 2002. A molecular phylogeny of anseriformes based on mitochondrial DNA analysis. Mol. Phylogenet. Evol. 23, 339–356] that Cape Barren goose is the sister taxon to coscoroba. The time of insertion was approximately 10.5 Mya or less estimated from mitochondrial DNA sequence information. Because this is a recent event, the DNA sequence of this CR1 should be close to that existing at the time of its insertion. This is reflected by the consistency of several structural features expected in a new CR1 copy such as the unaltered flanking target site duplication and inverted repeats that lie 22 bp apart near the 3′ end of the element. Hybridization experiments show that numerous copies of sequences closely related to the coscoroba CR1 element are dispersed throughout the genomes of tested Anseriformes, but none were detected in representatives of Galliformes and Struthioniformes.

Identification and characterization of a functional, alternatively spliced Toll‐like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens

Based upon the recognition of antiviral compounds and single stranded viral RNA the Toll-like receptors TLR7 and TLR8 are suggested to play a significant role in initiating antiviral immune responses. Here we report the molecular characterization of the chicken TLR7/8 loci which revealed an intact TLR7 gene and fragments of a TLR8-like gene with a 6-kilobase insertion containing chicken repeat 1 (CR1) retroviral-like insertion elements. The chicken TLR7 gene encodes a 1047-amino-acid protein with 62% identity to human TLR7 and a conserved pattern of predicted leucine-rich repeats. Highest levels of chicken TLR7 mRNA were detected in immune-related tissues and cells, especially the spleen, caecal, tonsil and splenic B cells. Alternative spliced forms of TLR7 mRNA were identified in chicken, mouse and human and expressed in similar tissues and cell types to the major form of chicken TLR7. The chicken TLR7+ HD11 cell line and fresh splenocytes produced elevated levels of interleukin-1beta (IL-1beta) mRNA after exposure to the agonists R848 and loxoribine. Interestingly, none of the TLR7 agonists stimulated increased type I interferon (IFN) mRNA whereas poly(I:C) (a TLR3 agonist) up-regulated both chicken IFN-alpha and chicken IFN-beta mRNA. In contrast, TLR7 agonists, particularly R848 and poly(U) stimulated up-regulation of chicken IL-1beta, and chicken IL-8 mRNAs more effectively than poly(I:C). Stimulation of chicken TLR7 with R848 was chloroquine sensitive, suggesting signalling within an endosomal compartment, as for mammalian TLR7. The deletion of TLR8 in galliforms, accompanied with the differential response after exposure to TLR7 agonists, offers insight into the evolution of vertebrate TLR function.

Fine tuning the structure of the Cu2+ complex with the prion protein chicken repeat by proline isomerization

The interaction between the single hexarepeat unit of chicken prion protein [ChPrP(54-59)] and Cu(II) was investigated by NMR, finding different coordination modes for the trans/trans and cis/trans isomers.

NfCR1, the first non‐LTR retrotransposon characterized in the Australian lungfish genome, Neoceratodus forsteri, shows similarities to CR1‐like elements

The genomes of lungfish, together with those of some urodele amphibians, are the largest of all vertebrate genomes. It has been assumed that the bulk of the DNA making up these large genomes has been derived from repeat elements, like the noncoding DNA of those genomes that have been sequenced (e.g., human). In an attempt to characterize repeat sequences in the lungfish genome, we have isolated, by restriction enzyme digestion of genomic DNA, sequences of a repeat element in Neoceratodus forsteri, the most primitive of the living lungfishes. The fragments sequenced from the EcoRI and BglII digests were used to perform genome walking PCR in order to obtain the full sequence of the repeat element. This element shares homology with the non-LTR (LINE) element, Chicken Repeat 1 (CR1), described for several vertebrates and some invertebrates; we have called it N. forsteri CR1 (NfCR1). NfCR1 shares all the domains of other CR1 elements but it also has several unique features that suggest it may no longer be active in the lungfish genome. It occurs in both full-length and 5'-truncated versions and in its present "inactive" form represents approximately 0.05% of the lungfish genome.