The longest 18S ribosomal RNA ever known

Abstract

An EMBL4 recombinant phage which encodes one of the full length of the aphid ribosomal DNA has been isolated from the aphid genomic library. Determination of the complete nucleotide sequence of the aphid 18s rRNA gene revealed that it is 2469 bp with a G + C content of 59%. The aphid 18s rRNA gene studied here is the longest and has the highest G + C content among the 18s rRNA genes examined so far. Evidence provided by the S1 nuclease assay suggests that the aphid 18s rRNA gene examined in this study is not a pseudogene containing an insertion sequence. Based on the nucleotide sequence of the 18s rRNA gene, we constructed a presumed secondary-structure model of the aphid 18s rRNA. In the aphid 18s rRNA, the eucaryote-specific E21 and 41 region are supposed to be longer and more complex than the counterparts of other 18s rRNA.It is generally known that rRNA share high similarity among various species. The small ribosomal subunit RNA (18s rRNA), which are encoded by nuclear genomes, have lengths close to 1800 bp and share significant nucleotide sequence identity with similar secondary structures. So far, 62 complete 18s rRNA sequences of eucaryotes, among which six vertebrates, four arthropods and one nematode are included, have been published [l] and extensively used as molecular clocks for evolutionary studies. Previously, we suggested that the aphid 18s rRNA has a molecular mass significantly larger than those of other eucaryotic 18s rRNA, suggesting that this exceptional structure may provide a clue to understand the general functions of the rRNA in the eucaryotic ribosomes To study the exceptional structure of this molecule further, we have cloned the aphid 18s rRNA gene and determined its entire nucleotide sequence. Also, based on the result we have presumed its secondary structure taking special note of the eucaryote-specific E21 region. 121. MATERIALS AND METHODS MaterialsA long-established parthenogenetic clone of the pea aphid, Acyrthosiphon pisum (Harris) was reared on young broadbean plants, Viciujaba L, at 15 "C in a short-day regime with a 12-h photo period. The collected aphids were stored at -80 "C until use. DNA isolation and cloningHigh-molecular-mass genomic DNA was isolated from the aphids by the method described [ 3 ] . EcoRI partial digests ofCorrespondence fo H. Ishikawa, Zoological Institute, Faculty of Science, University of Tokyo, Hongo 7-3-1, Tokyo, Japan 113 aphid DNA were ligated into EMBL4 at EcoRI sites using T4 ligase, packaged as described [4] and introduced into Escherichia coli LE392. The genomic library was screened [5] by the plaque-hybridization method using fragments from Bombyx mori ribosomal DNA as probes [6]. Subcloning and sequencingIsolation of bacteriophage DNA, subcloning of the fragment into plasmid Bluescript SK' and KS' , transformation into E. coli XL1 Blue, plasmid growth, isolation of plasmid DNA and restriction enzyme digestions were performed according to the standard methods described [4]. Plasmid subclones of deletion series were constructed and sequenced b...

Molecular phylogeny of the homoptera: a paraphyletic taxon

“…The 18S genes of the Sternorrhyncha sequenced in this study clearly have experienced unique evolutionary events compared to Auchenorrhyncha, Hemiptera, and outgroup taxa. The extra sequence found in sternorrhynchans is consistent with the discovery that the 18S rDNA of the pea aphid (Acyrthosiphon pisum) is almost 500 base pairs longer than that of most other insects (Kwon et al 1991). In the pea aphid, the extra, GC-rich sequence is found mainly in three variable regions, including the V2.…”

18S rRNA Secondary Structure and Phylogenetic Position of Peloridiidae (Insecta, Hemiptera)

“…(Hendriks et al, 1988a); CrustaceaArtemia salina (Nelles et al, 1984); Insecta: Coleoptera-Tenebrio molitor (Hendriks et al, 1988b); Hemiptera-Acyrthosiphon pisum (Kwon et al, 1991); Diptera-Drosophila melanogaster (Hancock et al, 1988;Gutell, 1994;van de Peer et al, 2000).…”

Molecular Evolution of the Small Subunit Ribosomal DNA in Woodlice (Crustacea, Isopoda, Oniscidea) and Implications for Oniscidean Phylogeny

“…Some crustacean species have remarkably long ssu rDNA sequences (Ͼ2900 bp) (Crease and Colbourne, 1998;Held and Wä gele, 1998), as also found in some Protozoa (Hinkle et al, 1994), insects (Tautz et al, 1988;Kwon, 1991;Campbell et al, 1994;Chalwatzis et al, 1995), and a flatworm (Piçon et al, 1996). The unusual lengths are caused by hypervariable regions or expansion elements which have been proven useful for the examination of the relationships in the genus Cicindela (Coleoptera) (Vogler et al, 1997).…”