Full-length-enriched cDNA libraries from Echinoeoccus granulosus contain separate populations of oligo-capped and trans-spliced transcripts and a high level of predicted signal peptide sequences

被引:61
作者
Fernández, C
Gregory, WF
Loke, P
Maizels, RM
机构
[1] Univ Edinburgh, Inst Cell Anim & Populat Biol, Edinburgh EH9 3JT, Midlothian, Scotland
[2] Univ Republica, Fac Chem, Dept Immunol, Montevideo, Uruguay
基金
英国惠康基金;
关键词
cestode; genome; helminth; open reading frames; untranslated regions;
D O I
10.1016/S0166-6851(02)00098-1
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The tissue-dwelling larval stages of the cestode Echinococcus granulosus are intimately associated with the host, implying that a range of molecular mediators may be secreted by the parasite into the host environment. These mediators are being sought through a transcriptome-based analysis, Using recombinant cDNA libraries, Conventional cDNA libraries of E granulosus contain high levels of mitochondrial transcripts, as well as host (bovine) genomic DNA. In particular, 60%, of a conventional protoscolex stage cDNA library corresponds to the large subunit (LSU) of mitochondrial rRNA. We attribute the presence of LSU rRNA copies to its polyadenylation in E granulosus. To circumvent this problem. we adapted the 5' Rapid Amplification of cDNA Ends (RNA-ligase mediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5' end of full-length mRNA. By ligating a specific oligonucleotide (oligo-cap) to 7MG-bearing mRNA, three cDNA libraries were made by PCR from oligo-cap and oligo-dT primers. Analysis of these libraries showed that mitochondrial RNA contaminants had been excluded. Moreover, no bovine genomic sequences were detected. In parallel, we constructed three cDNA libraries using the newly described trans-spliced leader (SL) from Echinococcus. Although these represent a smaller subset of parasite genes, mitochondrial and genomic contributions were again excluded. In both cases, a majority of cDNAs (61-92%) were judged to contain the initiation ATG codon, and 11-27%, of inserts included potential N-terminal signal sequences. The 5' UTR tracts of most oligo-capped cDNAs were < 100 nt, although ∼8%, were longer than this. Among the trans -spliced cDNAs, 43% potentially utilise the AUG donated by the SL, and in only 6% was the SL separated from an endogenous putative start site by > 60 nt, Sequence analysis of randomly selected clones shows virtually no overlap between the oligo-capped and SL libraries, indicating that trans-spliced E granulosus mRNAs appear to be insensitive to the enzymatic treatments used to 'oligo-cap' unspliced mRNAs. The oligo-capped and SL strategies represent efficient and complementary pathways to isolate full-length cDNA clones from this cestode parasite and, possibly, from related parasitic flatworms, (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:171 / 180
页数:10
相关论文
共 43 条
[1]  
ALTSCHUL SF, 1990, J MOL BIOL, V215, P403, DOI 10.1006/jmbi.1990.9999
[2]   Presence of mitochondria-type ribosomes outside mitochondria in germ plasm of Drosophila embryos [J].
Amikura, R ;
Kashikawa, M ;
Nakamura, A ;
Kobayashi, S .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (16) :9133-9138
[3]  
BANGS JD, 1992, J BIOL CHEM, V267, P9805
[4]   MITOCHONDRIAL LARGE RIBOSOMAL-RNA - AN ABUNDANT POLYADENYLATED SEQUENCE IN DROSOPHILA [J].
BENKEL, BF ;
DUSCHESNAY, P ;
BOER, PH ;
GENEST, Y ;
HICKEY, DA .
NUCLEIC ACIDS RESEARCH, 1988, 16 (20) :9880-9880
[5]   MAJOR TRANSCRIPT OF THE FRAMESHIFTED COXLL GENE FROM TRYPANOSOME MITOCHONDRIA CONTAINS 4 NUCLEOTIDES THAT ARE NOT ENCODED IN THE DNA [J].
BENNE, R ;
VANDENBURG, J ;
BRAKENHOFF, JPJ ;
SLOOF, P ;
VANBOOM, JH ;
TROMP, MC .
CELL, 1986, 46 (06) :819-826
[6]   Nematode spliced leaders - Ubiquity, evolution and utility [J].
Blaxter, M ;
Liu, L .
INTERNATIONAL JOURNAL FOR PARASITOLOGY, 1996, 26 (10) :1025-1033
[7]   CIS AND TRANS MESSENGER-RNA SPLICING IN C-ELEGANS [J].
BLUMENTHAL, T ;
THOMAS, J .
TRENDS IN GENETICS, 1988, 4 (11) :305-308
[8]   mRNA trans-splicing in the human parasitic cestode Echinococcus multilocularis [J].
Brehm, K ;
Jensen, K ;
Frosch, M .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (49) :38311-38318
[9]   MOLECULAR-CLONING AND CHARACTERIZATION OF ACTIN GENES FROM ECHINOCOCCUS-GRANULOSUS [J].
DASILVA, CMD ;
FERREIRA, HB ;
PICON, M ;
GORFINKIEL, N ;
EHRLICH, R ;
ZAHA, A .
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1993, 60 (02) :209-220
[10]   RNA TRANSSPLICING IN FLATWORMS - ANALYSIS OF TRANS-SPLICED MESSENGER-RNAS AND GENES IN THE HUMAN PARASITE, SCHISTOSOMA-MANSONI [J].
DAVIS, RE ;
HARDWICK, C ;
TAVERNIER, P ;
HODGSON, S ;
SINGH, H .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (37) :21813-21819