Characterization of novel Alu- and tRNA-related SINEs from the tree shrew and evolutionary implications of their origins

We characterized two novel 7SL RNA-derived short interspersed nuclear element (SINE) families (Tu types I and II) and a novel tRNA-derived SINE family (Tu type III) from the tree shrew (Tupaia belangeri). Tu type I contains a monomer unit of a 7SL RNA-derived Alu-like sequence and a tRNA-derived region that includes internal RNA polymerase III promoters. Tu type II has a similar hybrid structure, although the monomer unit of the 7SL RNA-derived sequence is replaced by a dimer. Along with the primate Alu, the galago Alu type II, and the rodent B1, these two families represent the fourth and fifth 7SL RNA-derived SINE families to be identified. Furthermore, comparison of the Alu domains of Tu types I and II with those of other 7SL RNA-derived SINEs reveals that the nucleotides responsible for stabilization of the Alu domain have been conserved during evolution, providing the possibility that these conserved nucleotides play an indispensable role in retropositional activity. Evolutionary relationships among these 7SL RNA-derived SINE families, as well as phylogenetic relationships of their host species, are discussed.     

Wide distribution of short interspersed elements among eukaryotic genomes.

Most short interspersed elements (SINEs) in eukaryotic genomes originate from tRNA and have internal promoters for RNA polymerase III. The promoter contains two boxes (A and B) spaced by approximately 33 bp. We used oligonucleotide primers specific to these boxes to detect SINEs in the genomic DNA by polymerase chain reaction (PCR). Appropriate DNA fragments were revealed by PCR in 30 out of 35 eukaryotic species suggesting the wide distribution of SINEs. The PCR products were used for hybridization screening of genomic libraries which resulted in identification of four novel SINE families. The application of this approach is illustrated by discovery of a SINE family in the genome of the bat Myotis daubentoni. Members of this SINE family termed VES have an additional B-like box, a putative polyadenylation signal and RNA polymerase III terminator.     

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.     

Integration site preferences of the Alu family and similar repetitive DNA sequences.

Numerous flanking nucleotide sequences from two primate interspersed repetitive DNA families have been aligned to determine the integration site preferences of each repetitive family. This analysis indicates that both the human Alu and galago Monomer families were preferentially inserted into short d(A+T)-rich regions. Moreover, both primate repeat families demonstrated an orientation specific integration with respect to dA-rich sequences within the flanking direct repeats. These observations suggest that a common mechanism exists for the insertion of many repetitive DNA families into new genomic sites. A modified mechanism for site-specific integration of primate repetitive DNA sequences is provided which requires insertion into dA-rich sequences in the genome. This model is consistent with the observed relationship between galago Type II subfamilies suggesting that they have arisen not by mere mutation but by independent integration events.     

Species-specific homogeneity of the primate Alu family of repeated DNA sequences

We have determined the base sequence of several cloned Alu family members from the DNAs of a new world monkey (owl monkey) and a prosimian (galago). The three owl monkey Alu family members reported here belong to a single 300 base pair consensus sequence which closely resembles the human Alu family consensus. The galago Alu family members can best be represented as belonging to either of two related but distinct consensus sequences. One of the two galago Alu family subgroups (Type I) more accurately resembles the human consensus sequence than does the other subgroup (Type II). In this work we compare base sequences of human and galago Type I Alu family members. There are several examples of species-specific differences between the human and Type I galago sequences indicating that the Alu family members are effectively homogenized within a species.     

The human Alu SINE sequences - is there a role for selection in their evolution?

The Alu sequence is a SINE (Short INterspersed Element) that is abundant in the human genome. A new analysis⁽¹⁾ reveals an unexpected conservation of some bases in the DNA sequence of the element. The bases involved include those forming an RNA polymerase III promoter. An unresolved question is whether this conservation results from selection for transposability. This, in turn, is related to the larger question of the evolutionary relationship between members of the Alu sequence family.     

CetSINEs and AREs are not SINEs but are parts of cetartiodactyl L1

Short interspersed repetitive elements (SINEs) are widely distributed among the genomes of eukaryotes. We proposed previously that a SINE should be defined by the presence of a region homologous to a tRNA or to 7SL RNA, together with A-box and B-box promoter sequences, in order to distinguish SINEs from other short repetitive sequences, such as short segments of LINEs (long interspersed repetitive elements; Okada et al. Gene 205, 229–243, 1997). Numerous SINE sequences have been deposited to date in DNA databases. In some cases, however, designation of a particular sequence is problematic when the short repetitive sequence has been defined as a SINE without reference to the presence or absence of promoter elements specific for RNA polymerase III. We demonstrate here that four different sequences, namely, ARE1p, ARE2p, CetSINE1, and CetSINE2, each of which has been reported as a SINE, are, in fact, only partial sequences of members of a new subfamily of L1. We also demonstrate that members of this subfamily are distributed specifically among the genomes of cetartiodactyls. Received: 3 May 2000 / Accepted: 22 August 2000     

A novel class of SINE elements derived from 5S rRNA

Eukaryotic genomes are colonized by different retroposons, including short interspersed repetitive elements (SINEs). All currently known SINEs are derived from tRNA and 7SL RNA genes and exploit their type 2 internal pol III promoters. We report here a novel class of SINE elements, called SINE3, derived from 5S rRNA. SINE3s are transcribed from the type 1 internal pol III promoter. Approximately 10,000 copies of SINE3 elements are present in the zebrafish genome, they constitute approximately 0.4% of the genomic DNA. Some elements are as little as 1% diverged from each other, indicating that the retrotransposition of SINE3 in zebrafish is an ongoing process. The 3'-tail of SINE3 is significantly similar to that of CR1-like non-LTR retrotransposons, represented by numerous subfamilies in the zebrafish genome. Analogously to CR1-like elements, SINE3 copies are not flanked by target site duplications, and their 3' termini are composed of (ACATT)n and (ATT)n microsatellites, specific for different subfamilies of SINE3. Given the common structural features, it is highly likely that the enzymatic machinery encoded by CR1-like elements powers proliferation of SINE3.     

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.     

G-repeats: a novel hamster sine family.

A fragment of a hamster repetitive element inserted into the aprt locus of a radiation-induced mutant is a member of a novel interspersed repetitive (SINE) family constituting approximately 0.3 to 0.5% of the hamster genome (30 to 50,000 family members). Since this family was first detected in a gene rearranged after exposure to gamma irradiation, we have called these G-repeats. In common with other repetitive elements, members of this family are about 300 bp in length, are highly divergent (an average of 30% from the consensus), have an A + T rich sequence flanking one side, and can be found in short polydisperse circular (SPC) DNA. In contrast to some other families, G-repeats are not flanked by short direct repeats and lack sequences corresponding to the RNA polymerase III consensus promoter.     

SINEs

SINEs with internal promoters for RNA polymerase III are ubiquitous in the genomes of the animal kingdom, including invertebrates. Although the human Alu family, and related families, originates from 7SL RNA, all other SINEs originate from tRNA. SINEs have been amplified many times, altered in genomic organization and fixed in the population at certain stages of evolution. They can therefore be regarded as time-landmarks of evolution. It is proposed that both population genetics and molecular biology are required for understanding the expansion of SINEs.     

Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.      

Alu repeats in the human genome

Highly repetitive DNA sequences account for more than 50% of the human genome. The L1 and Alu families harbor the most common mammalian long (LINEs) and short (SINEs) interspersed elements. Alu elements are each a dimer of similar, but not identical, fragments of total size about 300 bp, and originate from the 7SL RNA gene. Each element contains a bipartite promoter for RNA polymerase III, a poly(A) tract located between the monomers, a 3'-terminal poly(A) tract, and numerous CpG islands, and is flanked by short direct repeats. Alu repeats comprise more than 10% of the human genome and are capable of retroposition. Possibly, these elements played an important part in genome evolution. Insertion of an Alu element into a functionally important genome region or other Alu-dependent alterations of gene functions cause various hereditary disorders and are probably associated with carcinogenesis. In total, 14 Alu families differing in diagnostic mutations are known. Some of these, which are present in the human genome, are polymorphic and relatively recently inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

Identification of a short interspersed repeat in the Reticulitermes lucifugus (Isoptera Rhinotermitidae) genome.

A SINE element (called Talua) has been isolated from Reticulitermes lucifugus genome, by means of sequence comparison between clones obtained through genomic restriction and aspecific PCR amplification. It posses all the structural features commonly found in short interspersed elements: (i) a RNA polymerase III internal promoter, (ii) flanking short direct repeats and (iii) a poly (A) tail. BLAST search reveals significant homology with other previously described SINEs and tRNAs. The repeats are G+C-rich, but they are located in A+T-rich regions. This biased genomic distribution results from the analysis of adjacent regions. A Talua element was also found in a microsatellite-containing clone from Cryptotermes secundus. The presence of the SINE also in the Kalotermitidae family, suggests the usefulness of Talua as a taxonomic marker at the family level. The importance of this element on termite genome evolution is discussed.     

Can SINEs: a family of tRNA-derived retroposons specific to the superfamily Canoidea.

A repetitive element of approximately 200 bp was cloned from harbour seal (Phoca vitulina concolour) genomic DNA. The sequence of the element revealed putative RNA polymerase III control boxes, a poly A tail and direct terminal repeats characteristic of SINEs. Sequence and secondary structural similarities suggest that the SINE is derived from a tRNA, possibly tRNA-alanine. Southern blot analysis indicated that the element is predominately dispersed in unique regions of the seal genome, but may also be present in other repetitive sequences, such as tandemly arrayed satellite DNA. Based on slot-blot hybridization analysis, we estimate that 1.3 x 10(6) copies of the SINE are present in the harbour seal genome; SINE copy number based on the number of clones isolated from a size-selected library, however, is an order of magnitude lower (1-3 x 10(5) copies), an estimate consistent with the abundance of SINEs in other mammalian genomes. Database searches found similar sequences have been isolated from dog (Canis familiaris) and mink (Mustela vison). These, and the seal SINE sequences are characterized by an internal CT dinucleotide microsatellite in the tRNA-unrelated region. Hybridization of genomic DNA from representative species of a wide range of mammalian orders to an oligonucleotide (30mer) probe complementary to a conserved region of the SINE confirmed that the element is unique to carnivores of the superfamily Canoidea.     

Birth of a gene: locus of neuronal BC200 snmRNA in three prosimians and human BC200 pseudogenes as archives of change in the Anthropoidea lineage

The gene encoding brain-specific dendritic BC200 small non-messenger RNA is limited to the primate order and arose from a monomeric Alu element. It is present and neuronally expressed in all Anthropoidea examined. By comparing the human sequence of about 13.2 kb with each of the prosimian (lemur 14.6 kb, galago 12 kb, and tarsier 13.8 kb) orthologous loci, we could establish that the BC200 RNA gene is absent from the prosimian lineages. In Strepsirhini (lemurs and lorises), a dimeric AluJ-like element integrated very close to the BC200 insertion point, while the corresponding tarsier region is devoid of any repetitive element. Consequently, insertion of the Alu monomer that gave rise to the BC200 RNA gene must have occurred after the anthropoid lineage diverged from the prosimian lineage(s). Shared insertions of other repetitive elements favor proximity of simians and tarsiers in support of their grouping into Haplorhini and the omomyid hypothesis. On the other hand, the nucleotide sequences in the segment that is available for comparison in all four species reveal less exchanges between Strepsirhini (lemur and galago) and human than between tarsier and human. Our data imply that the early activity of dimeric Alu sequences must have been concurrent with the activity of monomeric Alu elements that persisted longer than is usually thought. As BC200 RNA gave rise to more than 200 pseudogenes, we used their consensus sequence variations as a molecular archive recording the BC200 RNA sequence changes in the anthropoid lineage leading to Homo sapiens and timed these alterations over the past 35-55 million years.