Unique structure in the intergenic and 5′ external transcribed spacer of the ribosomal RNA gene from the pea aphid Acyrthosiphon pisum

Abstract

We analyzed the DNA sequence of the 5' external transcribed spacer (ETS) and part of the intergenic transcribed spacer (IGS) of the aphid ribosomal RNA gene (rDNA). The 5' ETS of aphid rDNA consists of 843 nucleotides with a G/C content of 69 mo1/100 mol, far higher than that of any other known 5' ETS for insect rDNA. The IGS of aphid rDNA contained a characteristic array of repeated sequences of 247 nucleotides. The repeated sequences were identical. It was shown that the number of the repeating sequence is heterogeneous.In most eukaryotic organisms, ribosomal RNA genes (rDNA) are encoded in multiple copies and the 18S, 5.8s and 28s rRNA-coding regions are transcribed as a larger precursor (pre-rRNA) by RNA polymerase I, which undergoes a series of post-transcriptional processing steps yielding the 18S, 5.8s and 28s rRNA found in ribosomes [l]. The cis-regulatory sequence of transcription initiation of rDNA by RNA polymerase I is apparently species specific, and those for the rDNA of many animals, including human In the present study, we have analyzed the DNA sequence of the 5' external transcribed spacer (ETS) and part of the intergenic transcribed spacer (IGS) of aphid rDNA and suggested the presence of an array of characteristic repetitive sequences in IGS that may be involved in the transcriptioninitiation mechanism of this gene. MATERIALS AND METHODS MaterialsA long-established parthenogenetic clone of pea aphid, Acyrthosiphon pisum (Harris) was maintained on young broad-bean plants, Vicia faba L, at 15°C in a regime with a 12-h photo period. The collected aphids were stored at -80°C until use. DNA isolation, cloning, subcloning and sequencingHigh-molecular-mass genomic DNA was isolated from aphids by the method described in [6]. The genomic library was constructed by insertion of partial EcoRI digest of peaaphid DNA into the EMBL4 phage, as described in [7]. tDNA was screened by the plaque-hybridization method, using fragments of Boinbyx mori rDNA as probes [8]. An aphid rDNA clone, lApR8, was digested with XhoIIXbaI; the resulting fragments, encoding most of the IGS region, were subcloned into the same sites of the plasmid Bluescript SK'. Plasmid subclones of deletion series were constructed using a deletion kit (Takara Shuzo Co., Japan) and sequenced by the dideoxychain-termination method [9] using the Sequenase version 2.0 kit (U. S. Biochem. Co.). Sequence arrangement and dotmatrix analysis were performed using the DNASIS V6.00 program (Hi tachi Software Engineering Co., Japan). RNA isolation and S1-nuclease-protection assayTotal cellular RNA for S1-nuclease mapping was prepared by the method described in [lo]. For the Sl-nuclease-protection assay, 5' 32P end-labeling of DNA fragments, using hybridization conditions of end-labeled DNA fragments with total cellular RNA, was performed according to the method described in [ll]. Southern hybridization analysisPea-aphid genomic DNA and subcloned rDNA, pApR868, were partially or completely digested with restriction enzymes, and the resulting fragments were ...

Nucleotide sequence and presumed secondary structure of the 28S rRNA of pea aphid implication for diversification of insect rRNA

“…We have already sequenced a larger part of the IGS of pea aphid rDNA, which, it turned out, contained a characteristic array of perfect repeats of 247 nucleotides (Kwon and Ishikawa 1992a). Therefore, our present determination of nucleotide sequence upstream of the repeating sequences in the IGS completed the sequencing of the IGS, and thus, an entire unit of the pea aphid rDNA (data not shown).…”

“…Therefore, our present determination of nucleotide sequence upstream of the repeating sequences in the IGS completed the sequencing of the IGS, and thus, an entire unit of the pea aphid rDNA (data not shown). Table 1 summarizes the length and G + C contents of regions of the aphid rDNA unit (Kwon and Ishikawa 1992a). Table 1 represents the data of kApR8 in which the IGS contains 11 perfect repeats of 247 nucleotides.…”

Ribosomal spacer length variability in the large raspberry aphid, Amphorophora idaei (Aphidinae: Macrosiphini)

“…The two smaller fragments (7-7.8 kb and 2.7-5.6 kb) result from rDNA repeats which contain an extra Bglll site within the IGS, cutting the larger fragment in two (Fenton et a/., 1994). Size variation in all three fragments is located within the IGS (Fenton et a/., 1994) and probably results from changes in copy number of tandemly repeated elements (Kwon & Ishikawa, 1992). Fragments located at each end of the array were not detected.…”

Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes ofApis mellifera(Insecta: Hymenoptera): structure, organization, and retrotransposable elements

“…It is difficult to predict the composition and structure of the internal region of the IGS flanked by SRp1 and SRp2 for two reasons. First, the number of SRp regions vary in arthropods, ranging from one in the pea aphid (Kwon & Ishikawa, 1992) to three in D. melanogaster (Tautz et al ., 1988). Thus, although we identified two distinct SRp regions in honey bee IGS, a third (or more) SRp region may exist in the central portion of the spacer.…”