Chicken Repeat Research Articles

Genomic Organization of the Chicken T-Cell Receptor .BETA. Chain D-J-C Region

We examined overlapping genomic clones containing the chicken T cell receptor (TCR) Dbeta-Jbeta-Cbeta complex, which contains a single diversity segment, four joining segments and four exons that encode the constant region. This sequence comprised 18.3 kb. All four Jbeta sequences possessed typical recombination signal sequences (RSS) with intervening 12-bp spacers at their 5'-ends and splice sites at their 3'-ends. No Jbeta-pseudogenes were identified. TGTG sequences in the RSS heptamer sequences were well conserved, as is the case in mammals. A chicken repeat 1-like sequence was found in the intron region between Jbeta-1336 and Cbeta, and several small repeat sequences were identified in intron regions throughout this cloned genome. As germline sequences revealed complete Jbeta sequences, the CDR3 (complementarity-determining region) sequences of TCRbeta from non-immunized splenocytes were analyzed. Non-coding (N) and palindromic (P) nucleotides were frequently observed at the Dbeta-Jbeta recombination sites. There were differences in length of deletion at the 5'-end of each Jbeta. Deletion of the 5'-end of Jbeta-1280 was particularly short when compared with that of Jbeta-1336, but there were no changes in the length of the CDR3 using any of the four Jbeta sequences.

Analysis of part of the chicken Rfp-Y region reveals two novel lectin genes, the first complete genomic sequence of a class I alpha-chain gene, a truncated class II beta-chain gene, and a large CR1 repeat.

The Rfp-Y region lies on the same microchromosome as the B-F/B-L region of the B complex, yet in contrast to the latter it is poorly characterised. To date it has been shown to contain at least two class I alpha-chain ( Y-F) genes, a class II B-chain gene and a C-type lectin-like gene. We describe the sequencing and analysis of some 20 kb of the Rfp-Y region, and identify several new genes. These include two novel C-type lectin-like genes ( Y-Lec1 and Y-Lec2) that differ strongly from the previously described C-type lectin-like gene found in the Rfp-Y region. We describe a complete genomic sequence of a class I alpha-chain ( Y-F) gene and its promoter from the Rfp-Y region. The predicted cDNA from this gene has high homology to the previously reported Y-F cDNAs. The promoter contains an altered enhancer A element. This portion of the Rfp-Y region also contains a truncated class II B-chain ( Y-LB) gene, as well as a large chicken repeat 1 (CR1) element.

Pido, a non-long terminal repeat retrotransposon of the chicken repeat 1 family from the genome of the Oriental blood fluke, Schistosomajaponicum

A newly described non-long terminal repeat (non-LTR) retrotransposon element was isolated from the genome of the Oriental schistosome, Schistosomajaponicum. At least 1000 partial copies of the element, which was named pido, were dispersed throughout the genome of S. japonicum. As is usual with non-LTR retrotransposons, it is expected that many pido elements will be 5′-truncated. A consensus sequence of 3564 bp of the truncated pido element was assembled from several genomic fragments that contained pido-hybridizing sequences. The sequence encoded part of the first open reading frame (ORF), the entire second ORF and, at its 3′-terminus, a tandemly repetitive, A-rich (TA6TA5TA8) tail. The ORF1 of pido encoded a nucleic acid binding protein and ORF2 encoded a retroviral-like polyprotein that included apurinic/apyrimidinic endonuclease (EN) and reverse transcriptase (RT) domains, in that order. Based on its sequence and structure, and phylogenetic analyses of both the RT and EN domains, pido belongs to the chicken repeat 1 (CR1)-like lineage of elements known from the chicken, turtle, puffer fish, mosquitoes and other taxa. pido shared equal similarity with CR1 from chicken, an uncharacterized retrotransposon from Caenorhabditiselegans and SR1 (a non-LTR retrotransposon) from the related blood fluke Schistosomamansoni; the level of similarity between pido and SR1 indicated that these two schistosome retrotransposons were related but not orthologous. The findings indicate that schistosomes have been colonized by at least two discrete CR1-like elements. Whereas pido did not appear to have a tight target site specificity, at least one copy of pido has inserted into the 3′-untranslated region of a protein-encoding gene (GenBank AW736757) of as yet unknown identity. mRNA encoding the RT of pido was detected by reverse transcription–polymerase chain reaction in the egg, miracidium and adult developmental stages of S. japonicum, indicating that the RT domain was transcribed and suggesting that pido was replicating actively and mobile within the S. japonicum genome.

A comparative analysis of invaded sequences from group IA phospholipase A2 genes provides evidence about the divergence period of genes groups and snake families

Two phospholipase A2 (PLA2) genes classified into group IA were cloned from the genomic library of the sea snake Laticauda semifasciata. Eight clones were obtained by PCR cloning procedure from genomic DNA of Laticauda laticaudata (four clones) and Laticauda colubrina (four clones). The genes were 3.6–4.4kbp in length. Intron and exon organization of the group IA PLA2 genes was the same as that of Naja sputatrix group IA PLA2 genes (four exons and three introns). There were two kinds of repetitive sequences in the first and second introns of all sequenced PLA2 genes. The differences in the length of these genes were derived from the length of their repetitive sequences. The chicken repeat-1 (CR1)-like long interspersed repeated DNA (LINE) sequences, different from the above repetitive sequences, were also found in all sequenced Laticauda PLA2 genes. A comparative analysis of groups IA, IA′ and IIA PLA2s genes suggests a period of CR1-like LINE integration during molecular and family evolution. The integration of CR1-like LINE into PLA2 genes occurred after the divergence of groups I and II PLA2s but before the divergence of groups, IA and IA′ PLA2s. These integration events occurred before the family divergence of Naja and Laticauda. The presence of CR1-like LINE and a comparison of intron and exon organization showed that the divergence of Naja and Bungarus occurred before the divergence of Laticauda and Naja.

Songbird Genomics: Analysis of 45 kb Upstream of a Polymorphic Mhc Class II Gene in Red-Winged Blackbirds (Agelaius phoeniceus)

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative γ-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.

A LINE element from the pufferfish (fugu) Fugu rubripes which shows similarity to the CR1 family of non-LTR retrotransposons.

This study describes the consensus sequence of a full-length (4585bp) non-LTR retrotransposon from the fugu fish, Fugu rubripes. The retrotransposon, termed Maui, is represented by a group of very similar LINE elements found as multiple copies within the fish genome. Two long open reading frames (ORFs) are predicted from the sequence. The first ORF has a domain resembling a novel zinc finger motif recently found in both a turtle and a chicken (CR1) non-LTR retrotransposon. The second ORF includes sequences homologous to the endonuclease, reverse transcriptase and carboxy-terminal domains found in other non-LTR retrotransposons. Sequence comparisons of the predicted translation products of the two ORFs indicate that Maui is most closely related to a class of non-LTR retrotransposons represented by the CR1-like elements (chicken repeat 1 elements) that are present in several avian species and have recently been described in the turtle Platemys spixii. The sequence of the 3' untranslated region also supports this relationship since Maui resembles the CR1 like elements in not having a poly-A tail.

Retrotransposable CR1-like elements in crotalinae snake genomes

A part of the 3′-flanking region of BP-II gene, which is one of Trimeresurus flavoviridis venom gland phospholopase A 2 (PLA 2) isozyme genes, has a region homologous to avian chicken repeat 1 (CR1)-element. In the present study, ten CR1-like elements were further identified in T. gramineus venom gland PLA 2 isozyme genes, T. flavoviridis PLA 2 inhibitor (PLI) genes, and T. flavoviridis and T. gramineus TATA-box binding protein (TBP) genes. Southern blot analysis using a probe for CR1 showed that Crotalinae snake genomes contain a number of CR1-like elements.

Chicken repeat 1 (CR1) elements, which define an ancient family of vertebrate non-LTR retrotransposons, contain two closely spaced open reading frames

Chicken repeat 1 (CR1) elements comprise a family of non-long terminal repeat (LTR) retrotransposons that have several noteworthy features. For example, whereas most other non-LTR elements have poly(A) tracts or other simple A-rich repeats at their 3′ ends, the 3′ ends of CR1 elements conform to the consensus [(CATTCTRT)(GATTCTRT) 1–3]. CR1 elements also display an unusual bias for severe 5′ truncations: only approx. 30 (out of a total of approx. 30 000) CR1 elements in the chicken genome include significant portions of the pol-like open reading frame (ORF) that we previously identified and partially sequenced [Burch et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8199–8203]. In the present study we derived a consensus sequence for this entire ORF (ORF2) as well as an upstream ORF (ORF1) and part of a 5′ untranslated region ( UTR). The conceptual translation product of ORF2 is predicted to contain an endonuclease domain in addition to a reverse transcriptase domain. These results suggest that CR1 elements retrotranspose using a “nick and prime” mechanism similar (but not identical) to other families of non-LTR elements.

A retrotransposon of the non-long terminal repeat class from the human blood fluke Schistosoma mansoni. Similarities to the chicken-repeat-1-like elements of vertebrates.

The genomes of representative species of fishes, amphibians, and reptiles contain non-long-terminal-repeat (non-LTR) retrotransposons showing strong sequence identity to the chicken repeat 1 (CR1) non-LTR retrotransposon from birds. These nonavian retroelements have been termed CR1-like elements. We have isolated sequences of a non-LTR retrotransposon from the human blood fluke Schistosoma mansoni. These schistosome sequences, which we have termed the SR1 family of non-LTR retrotransposons, contain regions of deduced amino acids characteristic of the CR1-like elements. SR1 elements possess atypical 3' termini consisting of the tandem repeat (AACCATTTG)2 which are similar in structure to the imperfect tandem repeat of the 3' termini of CR1. There are at least 200 copies of SR1 interspersed through the genome of S. mansoni. The structural and amino acid sequence similarities of SR1 with members of the CR1-like elements suggest that the SR1 family belongs to the CR1-like category of non-LTR retrotransposons. Although other non-LTR retrotransposons have been described in invertebrates, this is the first CR1-like element reported from a nonvertebrate taxon, suggesting that the phylogenetic distribution of CR1-like retrotransposons is not restricted to vertebrates.

Two chicken repeat one (CR1) elements lacking a silencer-like region upstream of the chicken avidin-related genes Avr4 and Avr5

Two repetitive elements of the chicken CR1 family, each located in the 5′ flanking region of the avidin-related genes Aur4 and Avr5, have been cloned and sequenced. Both elements are 721 by in length with 72% identity to a CR1 consensus sequence. They had a 191 bp deletion in a region corresponding to the functional silencer regions previously detected within the CR1 elements upstream of the chicken lysozyme and apo VLDLII genes.

Evolution of chicken repeat 1 (CR1) elements: evidence for ancient subfamilies and multiple progenitors.

Chicken repeat 1 (CR1) is an interspersed repetitive element that is a member of the non-long terminal repeat class of retrotransposons. A data set of chicken 95 CR1 elements was compiled and the phylogeny of the 52 elements with the most complete 3' ends was examined. We interpret the branching pattern as clustering into at least six subfamilies, designated A-F. The presence of highly similar elements within the B, C, D, and F subfamilies is evidence that a distinct progenitor has spawned each of these subfamilies. The nucleotide divergence between members of subfamily C was 5%-8%, suggesting that this subfamily has undergone a relatively recent burst of retrotransposition. The A and E subfamilies may have been spawned from ancestors of these four progenitors or from other, distinct progenitors. The consensus sequences for the six subfamilies showed considerable divergence, implying that the CR1 subfamilies are ancient. The CR1 elements in each subfamily have truncated 5' ends and a 3' end consisting of > or = 2 repeats of an 8-bp sequence. We estimate that there are approximately 100,000 CR1 elements in the chicken genome. Twelve CR1 sequences from avian species other than chicken were identified. Some of these sequences grouped into different subfamilies, demonstrating that multiple subfamilies existed early in avian evolution. Reptilian CR1 sequences were also identified, demonstrating that the CR1 element arose before the divergence of birds and reptiles.

Analysis of a CR1 (chicken repeat) sequence flanking the 5' end of the gene encoding α-skeletal actin

Genomic Southern blots of chicken DNA, probed with radiolabeled DNA fragments that flank the 5′ end of the asa gene encoding chicken α-skeletal actin, indicate the presence of repetitive nucleotide sequences. Sequence analysis of this region reveals a member of the CR1 family of middle-repetitive elements. A 186-bp restriction fragment carrying the 3′-end of this CR1 element binds factor(s) present in nuclear extracts, as assayed band-shift electrophoresis. However, the CR1 repeat has limited influence on transcription from the α-skeletal actin promoter, as assessed by CAT assays of transfected chicken myoblasts and fibroblasts.