Repetitive sequences of the tree shrew genome (Mammalia, Scandentia)

Copies of two repetitive elements of the genome of common tree shrew (Tupaia glis) were cloned and sequenced. The first element, Tu III, is a approximately 260 bp long short interspersed element (SINE) with the 5'-end derived from glycine RNA. Tu III carries long polypurine- and polypyrimidine-rich tracts, which may contribute to the specific secondary structure of Tu III RNA. This SINE was also found in the genome of smooth-tailed tree shrew of another genus (Dendrogale). Tu III seems to be confined to the order Scandentia (tree shrews) since it was not found in DNA of other tested mammals. The second element Tu-SAT1 is a tandem repeat with a monomer length of 365 bp. Some properties of its nucleotide sequence suggest that Tu-SAT1 is a centromeric satellite.     

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.     

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.     

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.     

The lipoxygenase pathway of Tupaia belangeri representing Scandentia. Genomic multiplicity and functional characterization of the ALOX15 orthologs in the tree shrew

The tree shrew (Tupaia belangeri) is a rat-sized mammal, which is more closely related to humans than mice and rats. However, the use of tree shrew to explore the patho-mechanisms of human inflammatory disorders has been limited since nothing is known about eicosanoid metabolism in this mammalian species. Eicosanoids are important lipid mediators exhibiting pro- and anti-inflammatory activities, which are biosynthesized via lipoxygenase and cyclooxygenase pathways. When we searched the tree shrew genome for the presence of cyclooxygenase and lipoxygenase isoforms we found copies of functional COX1, COX2 and LOX genes. Interestingly, we identified four copies of ALOX15 genes, which encode for four structurally distinct ALOX15 orthologs (tupALOX15a-d). To explore the catalytic properties of these enzymes we expressed tupALOX15a and tupALOX15c as catalytically active proteins and characterized their enzymatic properties. As predicted by the Evolutionary Hypothesis of ALOX15 specificity we found that the two enzymes converted arachidonic acid predominantly to 12S-HETE and they also exhibited membrane oxygenase activities. However, their reaction kinetic properties (K_M for arachidonic acid and oxygen, T- and pH-dependence) and their substrate specificities were remarkably different. In contrast to mice and humans, tree shrew ALOX15 isoforms are highly expressed in the brain suggesting a role of these enzymes in cerebral function. The genomic multiplicity and the tissue expression patterns of tree shrew ALOX15 isoforms need to be considered when the results of in vivo inflammation studies obtained in this animal are translated into the human situation.     

Novel Hypovirulence-Associated RNA Mycovirus in the Plant-Pathogenic Fungus Botrytis cinerea: Molecular and Biological Characterization

Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A −1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The −1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.     

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.     

Molecular characterization,balancing selection,and genomic organization of the tree shrew (Tupaia belangeri) MHC class I gene

The major histocompatibility complex (MHC) class I genes play a pivotal role in the adaptive immune response among vertebrates. Accordingly, in numerous mammals the genomic structure and molecular characterization of MHC class I genes have been thoroughly investigated. To date, however, little is known about these genes in tree shrews, despite the increasingly popularity of its usage as an animal model. To address this deficiency, we analyzed the structure and characteristic of the tree shrew MHC class I genes (Tube-MHC I) and performed a comparative gene analysis of the tree shrew and other mammal species. We found that the full-length cDNA sequence of the tree shrew MHC class I is 1074 bp in length. The deduced peptide is composed of 357 amino acids containing a leader peptide, an α1 and α2 domain, an α3 domain, a transmembrane domain and a cytoplasmic domain. Among these peptides, the cysteines, CD8⁺ interaction and N-glycosylation sites are all well conserved. Furthermore, the genomic sequence of the tree shrew MHC class I gene was identified to be 3180 bp in length, containing 8 exons and 7 introns. In 21 MHC class I sequences, we conducted an extensive study of nucleotide substitutions. The results indicated that in the peptide binding region (PBR) the rate of non-synonymous substitutions (dN) to synonymous substitutions (dS) was greater than 1, suggesting balancing selection at the PBR. These findings provide valuable contributions in furthering our understanding of the structure, molecular polymorphism, and function of the MHC class I genes in tree shrews, further improving their utility as an animal model in biomedical research.     

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.     

Sequencing and expression of the second allele of the interleukin-1beta1 gene in rainbow trout (Oncorhynchus mykiss): identification of a novel SINE in the third intron

A lambda clone containing a rainbow trout IL-1beta1 gene was isolated by a PCR screening strategy from a genomic library cloned in lambda GEM-11, and an EcoRI fragment from this clone was fully sequenced, and contained 1680 bp 5'-flanking sequence, the whole IL-1beta1 gene open reading frame, and the 3'-flanking region with two potential poly A signals and poly A sites. This clone encoded a protein that shared 99.8% identity to the previously published trout IL-1beta1 cDNA sequence, with only three base substitutions. The main difference was that this clone had an additional complete HpaI SINE insertion in the 3rd intron making intron III 211 bp larger (834 bp via 623 bp). Thus this sequence was designated as allele B (Big intron III) of IL-1beta1 and the previously reported sequence as allele S (Short intron III). Three lines of evidence (allele specific PCR, cloning and sequencing, and direct sequencing of PCR products) revealed that allele B was constitutively expressed and could respond to stimulation with lipopolysaccharide or trout recombinant IL-1beta. Searching of the GenBank database with the HpaI SINE sequence resulted in three additional HpaI loci being identified in rainbow trout. Another SINE retroposition was also identified in the same intron of both alleles of IL-1beta1 by comparison with the trout IL-1beta2 gene. This novel SINE sequence, sharing high homology with the HpaI SINE at the 3'-end region, is present in EST databases of several species including human, mouse and fish. The consensus of this novel SINE shares 57 to 61% identities to tRNA-Leu from different species. Another older retroposition event in the same intron of IL-1beta1 has also been hypothesised, recognised as six adenines, that may function as a RNA polIII terminator. A model for the IL-1beta1 allele formation is proposed. Following the earliest retroposition into one of the two IL-1beta genes that resulted from a genome duplication in salmonids, the proper environment for successive PV SINE retroposition was created. A recent retroposition of the HpaI SINE in IL-1beta1 resulted in the formation of the two alleles of IL-1beta1. Examination of the SINEs insertion and their host gene microenvironments revealed that the SINE retroposition does not appear random, both in the site selection and the direction of insertion. The mechanism governing this outcome is discussed.     

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.     

Two Tandemly Arrayed Transfer-RNA-Derived SINEs of the Medaka (<Emphasis Type="BoldItalic">Oryzias latipes</Emphasis>)

: Two tandemly arrayed short interspersed repetitive element (SINE) sequences were found in medaka (Oryzias latipes). These two SINE sequences, designated SINE1 and SINE2, were flanked by a 180-bp AT-rich region. Both appeared to be derived from transfer RNA. The former exhibited 80% sequence homology to human tRNA^Ala and the latter exhibited 94% sequence homology to rat tRNA^Ser. SINE1 contained the retroviral U5 region, whereas SINE2 did not. This is the first sequence-level demonstration of the existence of neighboring SINEs in medaka.