GTMB 7 - Gene Therapy & Molecular Biology

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