GTMB 7 - Gene Therapy & Molecular Biology

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Almazán et al: Expressed sequence tags in Ixodes scapularis2E8 Esterase CD0525312F92F12Identity to AvGI TC255 (A.variegatum) & hypotheticalprotein FLJ12475 (H. sapiens)Transmembrane G-proteinresponsiveadenylyl cyclaseCD052532CD0525332G8 Lysyl-tRNA synthetase CD0525342H11Sodium- and chloride-dependenttaurine transporterCD0525353C12 RNA polymerase III CD0525363E1 Beta-adaptin CD0525373E2Microtubule-associated protein,RP/EB familyCD0525383E4 Myosin II regulatory light chain CD0525393E6UnknownZinc finger like proteinCD0525403E10 Mannose binding lectin (rhea) CD0525413E12 Clathrin heavy chain (Chc) CD0525423F4Identity to M. musculus adultmale testis cDNA3F10 Identity to D. melanogaster P-element somatic inhibitor (Psi)3G114A84A104A12Identity to D. melanogaster BM-40 extracellular basementmembrane proteinIdentity to D. melanogasterregulator of gene transcription(Chi)Identity to D. melanogasterhomeoprotein phtfAmino acid transporter system A(ATA2)CD052543CD052544CD052545CD052546CD052547CD0525484B2 Calmodulin CD0525494B7 Alpha-tubulin CD0525504C94C114D64D74E64D8Identity to D. melanogastertransducin (G protein)-likeenhancer of split 3, homolog ofE(spl)Intracellular receptor of activatedprotein kinase C1 (Rack1)Identity to D. melanogasterCG10395 cDNAIdentity to D. melanogasterLD23959 cDNAIdentity to D. melanogasterCG13597 cDNAIdentity to H. sapienshypothetical protein FLJ10342CD052551CD052552CD052553CD052554CD052555CD0525564E1 Pre-mRNA splicing factor CD0525574E3Receptor signaling proteinserine/threonine kinaseCD0525584F8 Nucleotidase CD0525594F1 Block of proliferation 1 (Bop1) CD0525604G14G2Identity to H. sapienshypothetical protein MGC2404LRP/alpha-2-macroglobulinreceptorCD052561CD0525624G5 Disulfide isomerase CD0525634G8 Fumarate hydratase CD0525644G10Rab3D (member of the Rassuperfamily of small GTPases)CD0525654G11 Chloride channel CD0525664H4 Solute carrier protein CD0525671B7 Mitochondrion NR1B8 Mitochondrion NR2E9 Mitochondrion NR2G11 Mitochondrion NR3C6 Mitochondrion NR3G4 Mitochondrion NR4A2 Mitochondrion NR4E9 Mitochondrion NR2A6 Mitochondrion NR4G7NAD-dependent malatedehydrogenaseNR3D4 Cytochrome c oxidase I (COI) NR1C2 Cytochrome c oxidase II (COII) NR4D2Cytochrome c oxidase III(COIII)NR1G4 Cytochrome b (cytb) NR2G9 16S ribosomal RNA NR1F42C73B64G124H2UnknownIdentity to I. scapularis cloneAC22 microsatellite sequence(AF331735)UnknownContains microsatellite sequenceUnknownContains a microsatellitesequenceUnknownContains microsatellite sequenceUnknownContains microsatellite sequenceCD052568CD052569CD052570CD052571CD052572NR, Not reported to the EST database for being identicalto mitochondrial sequencesThe majority of clones with gene ontology assignedcorresponded to non-nuclear gene products involved incell growth and maintenance, including genes with ligandbinding, carrier or enzymatic activities (Table 2).Seventeen clones contained sequences corresponding totick mitochondrion and were not submitted to the ESTdatabase. Other clones such as 2A9 and 1D6, althoughprobably coding for mitochondrial proteins, were analyzedand submitted to the EST database. Interestingly, 11clones encoded gene products localized in the cell nucleus(Table 2).The average G + C content of the EST dataset(47,503 bases excluding the poly-A tails with 171 (0.4%)undetermined nucleotide positions) was 54%, but somesequences, such as clone 2A9 which probably codes for amitochondrial protein, had only a 25% G + C content.46
Gene Therapy and Molecular Biology Vol 7, page 47Some short ESTs in clones 1D1 and 2D5 contained a longstretch of T.Vaccination of mice with plasmid DNA followed bytick infestation resulted in some cDNA clones that had aninhibitory effect on tick infestations, while others appearedto promote tick feeding (Table 3). The cDNAs inhibitingtick infestation were identical to nucleotidase, heat shockproteins, beta-adaptin, chloride channel, ribosomalproteins and proteins with unknown function. cDNAclones identical to beta-amyloid precursor, block ofproliferation, mannose-binding lectin, RNA polymeraseIII, ATPases and a protein of unknown function enhancedtick feeding.Further characterization of cDNAs that affectedlarval development (Table 3) was conducted for allclones except for 4D8, 4F8, 4D6 and 4E6, which producedhigh inhibition of tick infestation and are currently beingstudied separately as recombinant proteins expressed inEscherichia coli.The pool of heat shock proteins hsp70 and hsp60cDNAs conferred partial protection against tickinfestations and did not affect molting (Table 3). ThecDNA sequences for hsp70 and hsp60 in clones 1C10 and3F6, respectively, were partial and contained the regioncoding for the C-terminal of the protein, and were highlyidentical to other hsp70 sequences (data not shown).The sequence of hsp70 contained a 3’ untranslatedregion (UTR) of 299 bp before the poly-A tail. The clone3E1 contained a cDNA identical to the beta-adaptin thatproduced a 27% inhibition of tick infestation and a 5%inhibition of molting to the nymphal stage aftervaccination and tick challenge (Table 3). The completesequence was determined for the clone 3E1 (Figure 1A),and contained an insert of 1,942 bp encoding for apredicted protein of 191 amino acids. The sequence of thisprotein was shorter than that for other beta-adaptins(Figure 1B), suggesting that it could encode for a betaadaptinappendage or it may be a partial cDNA sequencebecause of a long 3’ UTR of 1,334 bp located before thepoly-A tail.Table 2. I. scapularis gene ontology assignments.Category Number of clones % of 87 clones with geneontology assignmentsCellular component% of 101 clones with identity toreported sequencesCell 32 36.78 31.88Mitochondria 17 15.54 16.83Cell membrane 14 16.09 13.86Nucleus 11 12.64 10.89Extracellular 2 2.30 1.98Unlocalized 2 2.30 1.98Unknown 9 10.34 8.91Biological processCell growth or maintenance 61 70.11 60.40Physiological process 8 9.20 7.92Developmental process 5 5.75 4.95Cell communication 2 2.30 1.98Unknown 11 12.64 10.89Molecular functionLigand binding or carrier 30 34.48 29.70Enzyme 29 33.33 28.71Transporter 9 10.34 8.91Chaperone 2 2.30 1.98Structural molecule 7 8.05 6.93Unknown 10 11.49 9.90Gene ontology assignments were made according to Ashburner et al. (2000) for non-redundant EST sequence data with the help ofGoFish v.1.0 (Berriz et al, 2003). The number of clone sequences falling into each category are listed and then calculated as a percent ofclones for which gene ontology was assigned and the total number of clones for which identity was found to previously publishedsequences.47
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