SINE Retrotransposition During the Evolution of the Pecoran Ruminants

SINE retrotransposition events have proven their value as phylogenetic markers in several eukaryotic taxa at different taxonomic levels. The genomes of ruminants contain three related SINE elements, Bov-tA, Bov-A2, and Bov-B. To estimate the time points of retrotransposition of individual copies of these SINEs, we designed PCR primers on database sequences containing SINE insertions in cattle, sheep, or goat genomes and tested for the presence of these copies in the genomes of other ruminants. It was checked by sequencing whether length variation of the PCR products reflected a SINE retrotransposition. One Bov-B and nine Bov-tA insertions were shared by cattle, sheep, goat, and giraffe, indicating an early retrotransposition event before the radiation of the Pecora, while three other Bov-tA and two Bov-B elements were apparently inserted later. The ruminant α-lactalbumine gene contains a hotspot of early and more recent Bov-tA insertions, a Bov-tA replacement as well as a recent Bov-B insertion. Three Bov-A2 insertions were found to be shared only by the Bovidae, the Bovini, and the Bos and Bison species, respectively, indicating that most Bov-A2 insertions are relatively recent. The time elapsed since the retrotransposition was also reflected in the degeneration of the direct repeats that flank SINE inserts. We suggest that retrotransposition of SINEs may serve as phylogenetic markers in the ruminant families, subfamilies, and even tribes. In addition, sequencing of SINE insertions revealed several other unique deletions/insertions that also may be informative for phylogenetic reconstructions of ruminants. Received: 19 January 2001 / Accepted: 6 June 2001     

Bov-B-mobilized SINEs in vertebrate genomes

Two new short retroposon families (SINEs) have been found in the genome of springhare Pedetes capensis (Rodentia). One of them, Ped-1, originated from 5S rRNA, while the other one, Ped-2, originated from tRNA-derived SINE ID. In contrast to most currently active mammalian SINEs mobilized by L1 long retrotransposon (LINE), Ped-1 and Ped-2 are mobilized by Bov-B, a LINE family of the widely distributed RTE clade. The 3' part of these SINEs originates from two sequences in the 5' and 3' regions of Bov-B. Such bipartite structure of the LINE-derived part has been revealed in all Bov-B-mobilized SINEs known to date (AfroSINE, Bov-tA, Mar-1, and Ped-1/2), which distinguishes them from other SINEs with only a 3' LINE-derived part. Structural analysis and the distribution of Bov-B LINEs and partner SINEs supports the horizontal transfer of Bov-B, while the SINEs emerged independently in lineages with this LINE.     

Conserved nucleotide differences and subfamily structure of porcine short interspersed elements

Interspersed elements are ubiquitous in the genomes of higher eukaryotes and account for over a third of the genomic DNA (Smit 1996). In swine the short interspersed elements, SINEs or PREs (porcine repetitive elements), have been found in a number of introns and 3' untranslated regions of different genes. However, compared to human Alu repeats the number of available PRE DNA sequences is still limited. In this study we have compared 85 PREs selected from DNA sequence database entries. The PREs were aligned and for each nucleotide position the relative frequencies of the four bases were calculated. A consensus sequence was derived from the first base usage. Similar to studies of SINEs in other species, the analysis showed that most mutations in PREs occur at CpG dinucleotide hot spots. The position variability for the two most frequent bases shows a bimodal distribution. The analysis suggests that the porcine SINEs can be divided into three major subfamilies sharing conserved nucleotide similarities.     

Single nucleotide polymorphisms (SNPs) within Bov-A2 SINE in the second intron of bovine and buffalo k-casein (CSN3) gene

The genetic polymorphism of an entire Bov-A2 element located in the second intron of the buffalo and bovine k-casein (CSN3) gene was investigated by amplification and sequencing of PCR products. Single nucleotide polymorphisms were detected. A PCR-RFLP method was developed to detect an A or G mutation at position 605 of bovine Bov-A2 element which creates a BfaI polymorphic site. The frequencies of the B allele, with the BfaI site, were for 0.275, 0.775, 0.750, 0.975, respectively, for Italian Holstein Friesian, Grey Alpine, Friuli Red Pied and Reggio bovine breeds. The mutation rate (substitutions and deletions/insertions per nucleotide site per year) was 2.5 x 10(-9) for Bov-A2 sequences in the second intron of CSN3. The comparison with other Bov-A2 elements suggests that this retroelement might be an important source of single nucleotide polymorphism for analysis of Bovidae genomes.     

Characterization of an abundant short interspersed nuclear element (SINE) present in Canis familiaris

A short interspersed nuclear element (Can SINE) of ∼130–150 bp was cloned and characterized from Canis familiaris. We demonstrate that this element is interspersed, present approximately every 5–8.3 kbp, and many are sufficiently close to allow IRS (interspersed repetitive DNA sequences) PCR. Sequence analysis of >20 Can SINEs from the dog has identified a conserved region that was used to design oligonucleotides for IRS PCR. Since Can SINEs are not present in human or rodent genomes, IRS PCR using oligonucleotides directed to the conserved region of Can SINEs can be used to simplify analysis of canid DNA in somatic cell hybrids, as well as in large insert cloning vectors. We demonstrate that the canid IRS products are polymorphic and could be developed as genetic markers for filter-based genotyping in this organism. Received: 23 July 1997 / Accepted: 9 September 1997     

Genealogy of families of SINEs in cetaceans and artiodactyls: the presence of a huge superfamily of tRNA(Glu)-derived families of SINEs.

Several novel (sub)families of SINEs were isolated from the genomes of cetaceans and artiodactyls, and their sequences were determined. From comparisons of diagnostic nucleotides among the short interspersed repetitive elements (SINEs) in these (sub)families, we were able to draw the following conclusions. (1) After the divergence of the suborder Tylopoda (camels), the CHRS family of SINEs was newly created from tRNA(Glu) in a common ancestor of the lineages of the Suina (pigs and peccaries), Ruminantia (cows and deer), and Cetacea (whales and dolphins). (2) After divergence of the Suina lineage, the CHR-1 SINE and the CHR-2 SINE were generated successively in a common ancestor of ruminants, hippopotamuses, and cetaceans. (3) In the Ruminantia lineage, the Bov-tA SINE was generated by recombination between the CHR-2 SINE and Bov-A. (4) In the Suina lineage, the CHRS-S SINE was generated from the CHRS SINE. (5) In this latter lineage, the PRE-1 family of SINEs was created by insertion of part of the gene for tRNA(Arg) into the 5' region of the CHRS-S family. The distribution of a particular family of SINEs among species of artiodactyls and cetaceans confirmed the most recent conclusion for paraphyly of the order Artiodactyla. The present study also revealed that a newly created tRNA(Glu)-derived family of SINEs was subjected both to recombination with different units and to duplication of an internal sequence within a SINE unit to generate, during evolution, a huge superfamily of tRNA(Glu)-related families of SINEs that are now found in the genomes of artiodactyls and cetaceans.     

Evolutionary History of B1 Retroposons in the Genus Mus

Short interspersed DNA elements (SINEs) amplify by retroposition either by (i) successive waves of amplification from one or a few evolving master genes or by (ii) the generation of new master genes that coexist with their progenitors. Individual, highly conserved, elements of the B1 SINE family were identified from the GenBank nucleotide database using various B1 subfamily consensus query sequences to determine their integration times into the mouse genome. A comparison of orthologous loci in various species of the genus Mus demonstrated that four subfamilies of B1 elements have been amplifying within the last 1–3 million years. Therefore, B1 sequences are generated by coexisting source genes. Additionally, three B1 subfamilies have been concurrently propagated during subspecies divergence and strain formation in Mus, indicating very recent activity of this retroposon family. The patterns of intra- and interspecies variations of orthologous loci demonstrate the usefulness of B1 integrations as a phylogenetic tool. A single inconsistency in the phylogenetic trends was depicted by the presence of a B1 insert in an orthologous locus exclusively in M. musculus and M. pahari. However, DNA sequence analysis revealed that these were independent integrations at the same genomic site. One highly conserved B1 element that integrated at least 4–6 million years ago suggests the possibility of occasional function for B1 integrations. Received: 25 February 2000 / Accepted: 5 June 2000     

Variable SINE 3′ poly(A) sequences: an abundant class of genetic markers in the pig genome

Previous studies on human DNA have shown that the 3 poly(A) tracts of Alu elements may display considerable genetic polymorphism. To explore whether this marker type is generally applicable in mammalian genomes, I analyzed porcine SINEs. A database screening revealed 17 porcine sequences with significant homology to a previously identified pig SINE. The occurrence in the database suggested a SINE frequency of one copy every 12 kb of pig DNA. All SINEs contained a 3 poly(A) tract with an average of 12 uninterrupted adenines. The repetitive regions were analyzed for polymorphism by locus-specific PCR amplification. Allelic length variation (two to five alleles among 10 pigs) was found at 8 out of 10 loci investigated, in most cases probably because of varying number of iterated adenine residues. There was a positive relationship between repeat length and the degree of polymorphism. Stable Mendelian inheritance was documented in 200 meioses each at four loci. The high genomic frequency of SINEs implies that a potentially informative marker may be found near any gene or in any cosmid clone. These SINE 3 poly(A) polymorphisms, termed SINEVA [SINE variable poly(A)s], thus provide an abundant and useful class of genetic marker in mammalian genomes.     

Definition,distribution, and use of a conserved Bovidae retroposon element sequence motif

Based on a data-base search, the sequences of 32 Bovidae retroposon elements have been compared. Two conserved areas are identified, and one of the corresponding sequences of the derived bovine consensus was used to design oligonucleotides as primer molecules for random DNA amplification of Bovidae DNA. Such a primer binding site should occur on average every 10,000 bp in the bovine genome, as suggested by a survey of published sequences. This estimate about the distribution of these possible primer binding sites was experimentally substantiated by mapping four of these primer binding sites within 40 kb of contiguous bovine DNA, carrying the heretofore undescribed bovine lactoferrin gene. Furthermore, these conserved, ubiquitous sequence motifs prove to be useful for mapping of bovine DNA.     

Genomic expansion of the Bov-A2 retroposon relating to phylogeny and breed management

Bov-A2 is a retroposon that is widely distributed among the genomes of ruminants (e.g., cow, deer, giraffe, pronghorn, musk deer, and chevrotain). This retroposon is composed of two monomers, called Bov-A units, which are joined by a linker sequence. The structure and origin of Bov-A2 has been well characterized but a genome-level exploration of this retroposon has not been implemented. In this study we performed an extensive search for Bov-A2 using all available genome sequence data on Bos taurus. We found unique Bov-A2-derived sequences that were longer than Bov-A2 due to amplification of three to six Bov-A units arranged in tandem. Detailed analysis of these elongated Bov-A2-derived sequences revealed that they originated through unequal crossing-over of Bov-A2. We found a large number of these elongated Bov-A2-derived sequences in cattle genomes, indicating that unequal crossing-over of Bov-A2 occurred very frequently. We found that this type of elongation is not observed in wild bovine and is therefore specific to the domesticated cattle genome. Furthermore, at specific loci, the number of Bov-A units was also polymorphic between alleles, implying that the elongation of Bov-A units might have occurred very recently. For these reasons, we speculate that genomic instability in bovine genomes can lead to extensive unequal crossing-over of Bov-A2 and levels of polymorphism might be generated in part by repeated outbreeding.     

Identification of a DNA methylation point in the promoter region of the bovine CYP21 gene

The CYP21 (steroid 21-hydroxylase) gene is involved in the synthesis of steroid hormones. Bov-A2 is a retroposon that is common in ruminant genomes. The promoter region of bovine CYP21 contains a short interspersed nucleotide element of Bov-A2, which overlaps a putative Sp1 binding site. We looked for RFLP/HpaII polymorphism in the Bov-A2 element in bovine Zebu breeds by PCR-RFLP, and examined whether polymorphism in this element is associated with methylation. Among DNA samples from 135 Brazilian Zebu breed cattle, we identified an RFLP/HpaII polymorphism (T/C), which, based on a restriction methylation-sensitive assay employing HpaII and isoschizomer MspI enzymes (methylation-sensitive and -non-sensitive enzymes, respectively), appears to be a DNA methylation point. This is the first report of this polymorphism and on DNA methylation in the bovine CYP21 promoter region in Brazilian Zebu cattle.     

Porcine SINE-associated microsatellite markers: evidence for new artiodactyl SINEs

Approximately 24% (170/710) of porcine (dG-dT)n·(dC-dA)n microsatellites isolated in our laboratory are associated with a previously described porcine Short Interdispersed Element (SINE) termed PRE-1 SINE. Another 5.6% (40/710) of the microsatellites were adjacent to two previously unidentified SINE sequences, which we have designated ARE-1P (Artiodactyl Repetitive Element-1 Porcine) and ARE-2P. The ARE repeats were also found in bovine microsatellite and genomic sequences in the GenBank database. Genotypic information was obtained from 68.9% of primers where at least one primer sequence was obtained from the PRE-1 SINE and 66.6% of primer pairs designed from the ARE SINEs. The use of primers derived from SINEs significantly increases the number of primer pairs available for genetic linkage studies in swine.     

Horizontal transfer of non-LTR retrotransposons in vertebrates

Since their discovery in family Bovidae (bovids), Bov-B LINEs, believed to be order-specific SINEs, have been found in all ruminants and recently also in Viperidae snakes. The distribution and the evolutionary relationships of Bov-B LINEs provide an indication of their origin and evolutionary dynamics in different species. The evolutionary origin of Bov-B LINE elements has been shown unequivocally to be in Squamata (squamates). The horizontal transfer of Bov-B LINE elements in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The direction of horizontal transfer from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution of Bov-B LINE elements. The ancestral snake lineage (Boidae) has been recognized as a possible donor of Bov-B LINE elements to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINE elements in Ruminantia and the fossil data of Ruminantia to be 40–50mya. The phylogenetic relationships of Bov-B LINE elements from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINE elements have been stably maintained by vertical transmission since the origin of Squamata in the Mesozoic era. This revised version was published online in July 2006 with corrections to the Cover Date.     

Barcoding bushmeat: molecular identification of Central African and South American harvested vertebrates

The creation and use of a globally available database of DNA sequences from a standardized gene region has been proposed as a tool for species identification, assessing genetic diversity and monitoring the legal and illegal trade in wildlife species. Here, we contribute to the Barcode of Life Data System and test whether a short region of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene would reliably distinguish among a suite of commonly hunted African and South American mammal and reptile species. We used universal primers to generate reference barcode sequences of 645 bp for 23 species from five vertebrate families (Crocodilidae, Alligatoridae, Bovidae, Suidae and Cercopithecidae). Primer cocktails yielded high quality barcode sequences for 179 out of 204 samples (87.7%) from all species included in the study. For most taxa, we sequenced multiple individuals to estimate intraspecific sequence variability and document fixed diagnostic characters for species identification. Polymorphism in the COX1 fragment was generally low (mean = 0.24%), while differences between congeneric species averaged 9.77%. Both fixed character differences and tree-based maximum likelihood distance methods unambiguously identified unknown and misidentified samples with a high degree of certainty. Barcode sequences also differentiated among newly identified lineages of African crocodiles and identified unusually high levels of genetic diversity in one species of African duiker. DNA barcoding offers promise as an effective tool for monitoring poaching and commercial trade in endangered species, especially when investigating semi-processed or morphologically indistinguishable wildlife products. We discuss additional benefits of barcoding to ecology and conservation.     

Evolution and recombination of bovine DNA repeats

The history of the abundant repeat elements in the bovine genome has been studied by comparative hybridization and PCR. The Bov-A and Bov-B SINE elements both emerged just after the divergence of the Camelidae and the true ruminants. A 31-bp subrepeat motif in satellites of the Bovidae species cattle, sheep, and goat is also present in Cervidae (deer) and apparently predates the Bovidae. However, the other components of the bovine satellites were amplified after the divergence of the cattle and the Caprinae (sheep and goat). A 23-bp motif, which as subrepeat of two major satellites occupies 5% of the cattle genome, emerged only after the split of the water buffalo and other cattle species. During the evolution of the Bovidae the satellite repeat units were shaped by recombination events involving subrepeats, other satellite components, and SINE elements. Differences in restriction sites of homologous satellites indicate a continuing rapid horizontal spread of new sequence variants. Correspondence to: J.A. Lenstra     

Porcine SINEs: Characterization and use in species-specific amplification

A porcine repetitive DNA sequence has been isolated from an intron of the glucose phosphate isomerase gene. The copy number of this and related sequences was estimated to be approximately 10(5) copies per genome. The sequence possesses all the characteristics of short interspersed elements (SINEs) described in other mammals: The repeat is 300 bp in length, has an poly(A)stretch, and contains insertion duplication sites. Homology to seven other porcine sequences, which also have the characteristics of SINEs, has been demonstrated. Primer oligonucleotides, based on conserved regions in the SINE sequences, have been synthesized. Using these primers, PCR-mediated specific amplification of porcine sequences was demonstrated from pig x mouse and pig x hamster hybrid cell lines. Cloning and sequencing of some amplified porcine sequences verify that the sites of priming are SINE sequences.     

Pstl repeat: a family of short interspersed nucleotide element (SINE)-like sequences in the genomes of cattle, goat, and buffalo.

The PstI family of elements are short, highly repetitive DNA sequences interspersed throughout the genome of the Bovidae. We have cloned and sequenced some members of the PstI family from cattle, goat, and buffalo. These elements are approximately 500 bp, have a copy number of 2 x 10(5) - 4 x 10(5), and comprise about 4% of the haploid genome. Studies of nucleotide sequence homology indicate that the buffalo and goat PstI repeats (type II) are similar types of short interspersed nucleotide element (SINE) sequences, but the cattle PstI repeat (type I) is considerably more divergent. Additionally, the goat PstI sequence showed significant sequence homology with bovine serine tRNA, and is therefore likely derived from serine tRNA. Interestingly, Southern hybridization suggests that both types of SINEs (I and II) are present in all the species of Bovidae. Dendrogram analysis indicates that cattle PstI SINE is similar to bovine Alu-like SINEs. Goat and buffalo SINEs formed a separate cluster, suggesting that these two types of SINEs evolved separately in the genome of the Bovidae.     

Characterization of a SINE species from vicuna and its distribution in animal species including the family Camelidae

Twenty-six sequences of a short interspersed repetitive element (SINE) with a size of approximately 150 base pairs (bp) were isolated from the genomic DNA of Vicugna vicugna (vicuna). RNA polymerase III split promoter sequence was observed in most of them, and many had direct repeats flanking to SINEs as well as a poly(A)-like structure. The SINE sequences were designated as ``vic-1' sequences. Comparison of the vic-1 consensus sequence with sequences registered in the DNA database (DDBJ/EMBL/GENBANK) revealed that the vic-1 sequence had a 79% homology with mouse ala-tRNA gene. In addition, the tRNA-related region of the consensus sequence was folded into a cloverleaf structure as with mouse ala-tRNA. These findings strongly indicated that vic-1 was a retroposon derived from ala-tRNA gene. The vic-1 sequences were used as a probe for dot-blot hybridization to examine the distribution of their homologous sequences in the genomes of various animal species spanning 14 orders, of which, homologous sequences were found only in the Camelidae family. In order to examine the phylogenetical relationship among vicuna, llama, and camel, vic-1 insertion analysis and homology analysis of vic-1 sequences were performed at each locus. The analyses indicated that vic-1 sequences were generated in a common ancestor of the animal species, and that camels first branched off from the clade Camelidae, followed by vicunas and llamas. Received: 5 July 2000 / Accepted: 14 November 2000     

Comparative and genetic analysis of the porcine glucocerebrosidase (GBA) gene

The genomic sequence of the porcine (Sus scrofa) glucocerebrosidase (GBA) gene (5.7 kb), encoding glucocerebrosidase (glucosylceramidase; acid beta-glucosidase; EC 3.2.1.45), was determined and compared with human (Homo sapiens) GBA and GBAP (pseudogene). The porcine gene harbours 11 exons and 10 introns, and the genomic organization is identical with human GBA. The exon sequences, coding for signal peptide and mature protein, show 81% and 90% sequence identity, respectively, with the corresponding human GBA sequences. Short interspersed elements, SINEs (PREs), are present in introns 2, 4 and 7. There is no evidence of a pseudogene in pig. The deduced protein sequence of GBA consists of 39 amino acids of signal peptide (long form) and 497 amino acids of the mature protein; the latter shows 90% sequence identity with the human protein. Four polymorphisms were observed within the porcine gene: insertion/deletion of one of the two SINEs (PREs) in intron 2 (locus PREA); deletion of a 37- to 39-bp stretch in intron 4 (one direct repeat and 5′ end of PRE); deletion of a 47-bp stretch in the middle part of PRE in intron 4 (locus PREB); and single-base transition (C–T) in intron 6 (locus HaeIII–RFLP). GBA was assigned to chromosome 4q21 by FISH and was localized to the same region by linkage analysis and RH mapping, i.e., to the chromosome 4 segment where quantitative trait loci for growth and some carcass traits are located.