Gene Ther Mol Biol Vol 7, 43-59, 2003

Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of expressed sequence tags

Research Article

Consuelo Almazan, Katherine M. Kocan, Douglas K. Bergman, Jose C. Garcia-Garcia, Edmour F. Blouin and José de la Fuente*

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.

__________________________________________________________________________________________

*Correspondence: José de la Fuente, Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078; Phone: (405) 744-0372; Fax: (405) 744-5275; e-mail: jose_delafuente@yahoo.com

Key words: tick, vaccine, tick cell culture, cDNA library immunization, EST, expression library immunization

Received: 23 May 2003; Accepted: 06 June 2003; electronically published: June 2003

Summary

Expression library immunization (ELI) combined with analysis of expressed sequence tags (ESTs) were used to identify genes transcribed in a cell line (IDE8) that was originally derived from embryos of Ixodes scapularis. A cDNA expression library was constructed from the IDE8 cells and cDNA clones were screened by ELI. Mice injected with cDNA clones were then infested with I. scapularis larvae. cDNA clones affecting larval feeding or development were subjected to single pass 5’ sequence analysis and the non-redundant sequences were putatively identified by sequence identity using the protein Basic Local Alignment Search Tool (BLAST) algorithm. Sequences of the clones were grouped according to the predicted function of the encoded proteins. 351 cDNAs that affected larval feeding and/or development were identified, of which 316 cDNA clones contained non-redundant sequences and 101 produced a significant identity to sequences reported previously. Gene ontologies could be assigned to 87 clones. Vaccination of mice with plasmid DNA followed by tick infestation resulted in identification of cDNA clones that inhibited tick infestation or promoted tick feeding. cDNAs that inhibited tick infestation were identical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins, and proteins with unknown function. cDNA clones that promoted tick feeding were identical to beta-amyloid precursor, block of proliferation, mannose-binding lectin, RNA polymerase III, ATPases and a protein of unknown function. Herein, we describe the sequence analysis of I. scapularis ESTs selected by ELI that affected larval tick feeding and/or development. These proteins may be useful for incorporation into vaccine preparations designed to interrupt the life cycle of I. scapularis and/or interfere with transmission of pathogens.

I. Introduction

Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important vector of pathogens in North America (Parola and Raoult, 2001). Ixodes spp. (Acari: Ixodidae) are distributed worldwide and are vectors of human pathogens, including Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic ehrlichiosis), Coxiella burnetti (Q fever), Francisella tularensis (tularemia), B. afzelii, B. lusitaniae, B. valaisiana and B. garinii, Rickettsia helvetica, R. japonica and R. australis, Babesia divergens, as well as tick-borne encephalitis (TBE) and Omsk Hemorrhagic fever viruses (Estrada-Peña and Jongejan, 1999; Parola and Raoult, 2001). Throughout eastern and southeastern United States and Canada, I. scapularis (the black legged tick) is the main vector of B. burgdorferi sensu stricto and A. phagocytophilum (Estrada-Peña and Jongejan, 1999; Parola and Raoult, 2001).

Control of tick infestations is difficult, particularly for multi-host ticks such as Ixodes spp. Presently, tick control is effected by integrated pest management in which different control methods are adapted in a geographic area against one tick species with due consideration to their environmental effects. Recently, development of vaccines against one-host Boophilus spp. has provided new possibilities for identification of protective antigens for use in vaccines for control of tick infestations (Willadsen, 1997; Willadsen and Jongejan, 1999; de la Fuente et al., 1999, 2000a; de Vos et al., 2001). Control of ticks by vaccination would avoid environmental contamination and selection of drug resistant ticks that can result from repeated acaricide application (de la Fuente et al., 1998; Garcia-Garcia et al., 1999). Anti-tick vaccines also allow for inclusion of multiple antigens in order to target a broad range of tick species, as well as pathogen-blocking antigens.

Development of high throughput DNA sequencing technologies and bioinformatic tools facilitate assignment of provisional function to expressed sequence tags (ESTs; Boguski et al., 1993). This approach has resulted in valuable information for the study of biological systems and for the identification of potential vaccine candidates (Lizotte-Waniewski et al., 2000; Knox et al., 2001; Tarleton and Kissinger, 2001; Touloukian et al., 2001; Kessler et al., 2002). In ticks, construction of EST databases has been reported for B. microplus (Crampton et al., 1998), Amblyomma americanum (Hill and Gutierrez, 2000) and A. variegatum (Nene et al., 2002). The application of EST technology has been used for characterization of gene expression in salivary glands of I. scapularis (Valenzuela et al., 2002), I. ricinus (Valenzuela, 2002), A. americanum and Dermacentor andersoni (Bior et al., 2002), for identification of genes differentially expressed in D. variabilis ovaries in response to rickettsial infection (Mulenga et al., 2003) and in I. ricinus salivary glands in response to blood feeding (Leboulle et al., 2002).

A new technique, expression library immunization (ELI), in combination with sequence analysis of ESTs, provides an alternative approach for identification of potential vaccine antigens that is based on rapid screening of the expressed genes without prior knowledge of the antigens encoded by the cDNAs. ELI was first reported for Mycoplasma pulmonis (Barry et al., 1995) and since then has been used for unicellular and multicellular pathogens and viruses (Manoutcharian et al., 1998; Alberti et al., 1998; Brayton et al., 1998; Melby et al., 2000; Smooker et al., 2000; Moore et al., 2001; Singh et al., 2002; Leclercq et al., 2003). Recently, we reported the first application of ELI to arthropods, specifically to I. scapularis (Almazán et al., 2003) in a mouse model system. A combination of cDNA ELI and EST analysis resulted in the selection of 351 cDNA clones affecting tick larval development (Almazán et al., 2003). After grouping the clones according to the putative function of predicted proteins, some cDNA pools resulted in the inhibition of tick infestation and others promoted tick feeding after ELI (Almazán et al., 2003).

Herein we describe the sequence analysis and characterization of I. scapularis ESTs that were identified by Almazán et al. (2003) using cDNA ELI and a mouse model for tick infestation.

II. Materials and Methods

A. Construction of the I. scapularis expression cDNA library.

The cDNA library was constructed from I. scapularis cultured embryonic IDE8 cells (Munderloh et al., 1994) as reported previously (Almazán et al., 2003). The expression library was constructed in the vector pEXP1 containing the strong human cytomegalovirus major immediate early promoter/enhancer (CMV_IE) (Clontech, Palo Alto, CA). The cDNA library contained 4.4 x 10⁶ independent clones and a titer of approximately 10¹⁰ cfu/ml with more than 93% of the clones with cDNA inserts. The average cDNA size was 1.7 kb (0.5-4.0 kb).

B. DNA vaccination and tick infestation.

Vaccinations with plasmid DNA and tick infestations were done as reported previously for the screening of the expression cDNA library by ELI using the mouse model of I. scapualris infestations (Almazán et al., 2003). Briefly, plasmid DNA was purified (Wizard SV 96 plasmid DNA purification system, Promega, Madison, WI) and used to inject CD-1 female mice, 5-6 weeks of age at the time of first vaccination. Mice were cared for in accordance with standards specified in the Guide for Care and Use of Laboratory Animals. Mice were injected using a 1 ml tuberculin syringe and a 27aG needle at days 0 and 14. Three to 6 mice per group were each immunized IM in the thigh with 1 µg total DNA/dose in 50 µl PBS. Control mice were injected with 1 µg vector DNA alone. Two weeks after the last immunization, mice were infested with 100 I. scapularis larvae per mouse. For tick infestations, mice were retrained in a small wire cage in a cardboard carton. One hundred larvae were counted and applied to the mice with a brush. Ticks were reared at the Oklahoma State University Tick Rearing Facility by feeding larvae on mice, nymphs on rabbits and adults on sheep. For these experiments, larvae were obtained from the eggs oviposited by sister females. Twelve hours after tick infestation, larvae in the bottom of the cage that did not attach were counted in order to calculate the number of attached larvae per mouse. Mice were then transferred to individual cages in which they were placed on an elevated 1/4” mesh wire platform over water (1/2” deep). Replete larvae dropping from each mouse were collected daily from the water and counted during 7 days. Time for larval development was evaluated from the day of tick infestation to the day in which the maximum number of replete larvae was collected. The inhibition of tick infestation (I) for each test group was calculated with respect to vector-immunized controls as [1-(RLn/RLc x RLic/RLin)] x 100, where RLn is the average number of replete larvae recovered per mouse for each test group, RLc is the average number of replete larvae recovered per mouse for control group, RLic is the average number of larvae attached per mouse for control group, and RLin is the average number of larvae attached per mouse for each test group. Engorged larvae were held in a 95% humidity chamber and allowed to molt. Molting of engorged larvae was evaluated 34 days after the last larval collection by visual examination of ticks under a dissecting light microscope. The inhibition of molting (M) for each test group was calculated with respect to controls as [1-(MLn/MLc x RLc/RLn)] x 100, where MLn is the average number of nymphs for each test group, MLc is the average number of nymphs for the control group, RLc is the average number of larvae recovered for the control group, and RLn is the average number of larvae recovered for each test group.

C. Plasmid DNA preparation and sequencing.

Bacterial colonies were inoculated in Luria-Bertani with 50 µg/ml ampicillin, grown for 16 hr in a 96-well plate and plasmid DNA purified (Wizard SV 96 plasmid DNA purification system, Promega, Madison, WI) and partially sequenced with a 5’ vector-specific primer (5’-CGACTCACTATAGGGAG-3’) at the Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University, using ABI Prism dye terminator cycle sequencing protocols developed by Applied Biosystems (Perkin-Elmer Corp., Foster City, CA). In most cases a sequence larger than 700 nucleotides was obtained.

D. Data analysis.

Nucleotide sequences were analyzed using the program AlignX (Vector NTI Suite V 5.5, InforMax, North Bethesda, MD). Multiple sequence alignment was performed using an engine based on the Clustal W algorithm (Thompson et al., 1994). Nucleotides were coded as unordered, discrete characters with five possible character-states; A, C, G, T, or N (missing) and gaps were coded as missing data. Phylogenetic trees were constructed based on a sequence distance method utilizing the Neighbor Joining algorithm of Saitou and Nei (1987). BLAST (Altschul et al., 1990) was used to search the NCBI databases to identify previously reported sequences with identity to those that we sequenced. Gene ontology assignments were made according to Ashburner et al. (2000) for non-redundant EST sequence data with the help of GoFish v.1.0 (Berriz et al., 2003).

III. Results

The screening of the I. scapularis expression cDNA library by ELI and EST analysis resulted in 351 cDNAs affecting larval development in the mouse model of tick infestation (Almazán et al., 2003). Of them, 316 cDNA clones contained non-redundant sequences and 101 (32%) produced a significant identity to previously reported sequences by BLAST analysis of NCBI nucleotide and protein databases (Table 1). Gene ontologies could be assigned to 87 clones (27.5% of non-redundant sequences and 86.1% of clones with identity to sequences reported previously) (Table 2).

Table 1. cDNA clones with identity to previously reported sequences.

EST clone	Predicted protein	GenBank accession number
1C11	Translation initiation factor 5A (eIF5A)	CD052489
1E6	Translation initiation factor 5C (eIF-5C)	CD052490
2D2	Initiate factor 5 (if5)	CD052491
1A10	Elongation factor 2	CD052492
4F7	Elongation factor 1alpha	CD052493
1F6	Ribosomal protein S4 (RpS4)	CD052494
2B8	Ribosomal protein S11 (RpS11)	NR
2F8	Laminin receptor 1 (ribosomal protein SA)	CD052496
2F10	Ribosomal protein L3 (RpL3)	NR
3A10	Ribosomal protein L7A (RpL7A)	CD052497
3G9	Ribosomal protein S8 (RpS8)	CD052495
3G10	Ribosomal protein L27A (RpL27A)	CD052498
3C3	QM homolog (DQM) ribosomal protein	CD052499
4D12	Proteasome/Signalosome subunit	CD052500
4E7	Proteasome subunit	CD052501
4D11	Proteasome subunit	CD052502
3D10	Ribophorin I	CD052503
1B12	Ubiquitin-conjugating enzyme	CD052504
1D10	Ubiquitin	CD052505
1A9	V-ATPase D subunit Contains microsatellite sequence	CD052506
1B2	V-ATPase C subunit	CD052507
EST clone	Predicted protein	GenBank accession number
4A4	V-ATPase E subunit	CD052508
1C5	Na+/K+ ATPase, alpha subunit	CD052509
2A9	NADH dehydrogenase	CD052510
1D6	NADH dehydrogenase subunit 5 (nad5)	CD052511
1A4	Aldehyde dehydrogenase	CD052512
1C8	Virilizer (vir)	CD052513
1C10	Hsp70	CD052514
3F6	Hsp60	CD052515
1D1	Nucleotide binding protein 1 (Nubp1)	CD052516
1D8	Identity to D. melanogaster GH03607 full length cDNA coding for a putative membrane protein	CD052517
1D11	Putative membrane protein	CD052518
1E7	Sterol carrier protein	CD052519
1F3	Cyclin C (CycC)	CD052520
3D9	Alpha tubulin	CD052521
2A7	Beta tubulin	CD052522
2A11	Notchless (Nle)	CD052523
2B2	Export factor binding protein 2 (Refbp2)	CD052524
2B7	G protein-coupled receptor	CD052525
2B9	Succinate dehydrogenase B (SdhB)	CD052526
2C12	Beta-amyloid precursor protein (APP)	CD052527
2D1	Fructose-1,6-bisphosphatase (fbp gene)	CD052528
2D5	DNA repair protein Rad1 (Rad1)	CD052529
2D6	Identity to S. pombe dim1+, helicase protein 1	CD052530
2E8	Esterase	CD052531
2F9	Identity to AvGI TC255 (A. variegatum) & hypothetical protein FLJ12475 (H. sapiens)	CD052532
2F12	Transmembrane G-protein-responsive adenylyl cyclase	CD052533
2G8	Lysyl-tRNA synthetase	CD052534
2H11	Sodium- and chloride-dependent taurine transporter	CD052535
3C12	RNA polymerase III	CD052536
3E1	Beta-adaptin	CD052537
3E2	Microtubule-associated protein, RP/EB family	CD052538
3E4	Myosin II regulatory light chain	CD052539
3E6	Unknown Zinc finger like protein	CD052540
3E10	Mannose binding lectin (rhea)	CD052541
3E12	Clathrin heavy chain (Chc)	CD052542
3F4	Identity to M. musculus adult male testis cDNA	CD052543
3F10	Identity to D. melanogaster P-element somatic inhibitor (Psi)	CD052544
3G11	Identity to D. melanogaster BM-40 extracellular basement membrane protein	CD052545
4A8	Identity to D. melanogaster regulator of gene transcription (Chi)	CD052546
4A10	Identity to D. melanogaster homeoprotein phtf	CD052547
4A12	Amino acid transporter system A (ATA2)	CD052548
4B2	Calmodulin	CD052549
4B7	Alpha-tubulin	CD052550
4C9	Identity to D. melanogaster transducin (G protein)-like enhancer of split 3, homolog of E(spl)	CD052551
4C11	Intracellular receptor of activated protein kinase C1 (Rack1)	CD052552
4D6	Identity to D. melanogaster CG10395 cDNA	CD052553
4D7	Identity to D. melanogaster LD23959 cDNA	CD052554
4E6	Identity to D. melanogaster CG13597 cDNA	CD052555
4D8	Identity to H. sapiens hypothetical protein FLJ10342	CD052556
4E1	Pre-mRNA splicing factor	CD052557
4E3	Receptor signaling protein serine/threonine kinase	CD052558
4F8	Nucleotidase	CD052559
4F1	Block of proliferation 1 (Bop1)	CD052560
4G1	Identity to H. sapiens hypothetical protein MGC2404	CD052561
4G2	LRP/alpha-2-macroglobulin receptor	CD052562
4G5	Disulfide isomerase	CD052563
4G8	Fumarate hydratase	CD052564
4G10	Rab3D (member of the Ras superfamily of small GTPases)	CD052565
4G11	Chloride channel	CD052566
4H4	Solute carrier protein	CD052567
1B7	Mitochondrion	NR
1B8	Mitochondrion	NR
2E9	Mitochondrion	NR
2G11	Mitochondrion	NR
3C6	Mitochondrion	NR
3G4	Mitochondrion	NR
4A2	Mitochondrion	NR
4E9	Mitochondrion	NR
2A6	Mitochondrion	NR
4G7	NAD-dependent malate dehydrogenase	NR
3D4	Cytochrome c oxidase I (COI)	NR
1C2	Cytochrome c oxidase II (COII)	NR
4D2	Cytochrome c oxidase III (COIII)	NR
1G4	Cytochrome b (cytb)	NR
2G9	16S ribosomal RNA	NR
1F4	Unknown Identity to I. scapularis clone AC22 microsatellite sequence (AF331735)	CD052568
2C7	Unknown Contains microsatellite sequence	CD052569
3B6	Unknown Contains a microsatellite sequence	CD052570
4G12	Unknown Contains microsatellite sequence	CD052571
4H2	Unknown Contains microsatellite sequence	CD052572

NR, Not reported to the EST database for being identical to mitochondrial sequences

The majority of clones with gene ontology assigned corresponded to non-nuclear gene products involved in cell growth and maintenance, including genes with ligand binding, carrier or enzymatic activities (Table 2). Seventeen clones contained sequences corresponding to tick mitochondrion and were not submitted to the EST database. Other clones such as 2A9 and 1D6, although probably coding for mitochondrial proteins, were analyzed and submitted to the EST database. Interestingly, 11 clones 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 some sequences, such as clone 2A9 which probably codes for a mitochondrial protein, had only a 25% G + C content. Some short ESTs in clones 1D1 and 2D5 contained a long stretch of T.

Vaccination of mice with plasmid DNA followed by tick infestation resulted in some cDNA clones that had an inhibitory effect on tick infestations, while others appeared to promote tick feeding (Table 3). The cDNAs inhibiting tick infestation were identical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins and proteins with unknown function. cDNA clones identical to beta-amyloid precursor, block of proliferation, mannose-binding lectin, RNA polymerase III, ATPases and a protein of unknown function enhanced tick feeding.

Further characterization of cDNAs that affected larval development (Table 3) was conducted for all clones except for 4D8, 4F8, 4D6 and 4E6, which produced high inhibition of tick infestation and are currently being studied separately as recombinant proteins expressed in Escherichia coli.

The pool of heat shock proteins hsp70 and hsp60 cDNAs conferred partial protection against tick infestations and did not affect molting (Table 3). The cDNA sequences for hsp70 and hsp60 in clones 1C10 and 3F6, respectively, were partial and contained the region coding for the C-terminal of the protein, and were highly identical to other hsp70 sequences (data not shown)

Table 2. I. scapularis gene ontology assignments.

Category	Number of clones	% of 87 clones with gene ontology assignments	% of 101 clones with identity to reported sequences
Cellular component
Cell	32	36.78	31.88
Mitochondria	17	15.54	16.83
Cell membrane	14	16.09	13.86
Nucleus	11	12.64	10.89
Extracellular	2	2.30	1.98
Unlocalized	2	2.30	1.98
Unknown	9	10.34	8.91
Biological process
Cell growth or maintenance	61	70.11	60.40
Physiological process	8	9.20	7.92
Developmental process	5	5.75	4.95
Cell communication	2	2.30	1.98
Unknown	11	12.64	10.89
Molecular function
Ligand binding or carrier	30	34.48	29.70
Enzyme	29	33.33	28.71
Transporter	9	10.34	8.91
Chaperone	2	2.30	1.98
Structural molecule	7	8.05	6.93
Unknown	10	11.49	9.90

Gene ontology assignments were made according to Ashburner et al. (2000) for non-redundant EST sequence data with the help of GoFish v.1.0 (Berriz et al., 2003). The number of clone sequences falling into each category are listed and then calculated as a percent of clones for which gene ontology was assigned and the total number of clones for which identity was found to previously published sequences.

Table 3. Summary of results of DNA vaccination and challenge with I. scapularis larvae in the mouse model of tick infestations.

EST cDNA clone	Predicted protein	Inhibition of tick infestation I (%)	Inhibition of molting M (%)
4D8	Identity to H. sapiens hypothetical protein FLJ10342 with unknown function	40 ^a	7 ^a
4F8	Nucleotidase	50^a	17^a
1C10^b	Hsp70	17^a	0^a
3F6^b	Hsp60
4D6	Identity to D. melanogaster CG10395 cDNA with unknown function	61	11
4E6	Identity to D. melanogaster CG13597 cDNA with unknown function	20	ND
3E1	Beta-adaptin	27	5
4G11	Chloride channel	38	30
17 clones^b	Ribosomal proteins	15^a	0^a
2C12	Beta-amyloid precursor protein (APP)	-8^c	ND
4F1	Block of proliferation Bop1	-39^c	ND
3E10	Mannose binding lectin	-48^a, c	ND
3C12^b	RNA polymerase III	-104^a, c	ND
2F9^b	Identity to A. variegatum AvGI TC255 & Homo sapiens hypothetical protein FLJ12475 with unknown functions
1A9, 1B2, 4A4^b	ATPase	-57^a, c	ND

^aData reported by Almazán et al. (2003). For all other experiments, mice were immunized with cDNA-containing expression plasmid DNA as described above. I and M were calculated as described in Materials and Methods section. ND, not determined.

^bPooled together for vaccination experiments by ELI (Almazán et al., 2003) (1C10 and 3F6, cDNA pool “Heat shock”; 3C12 and 2F9, cDNA pool “Secreted protein”; ribosomal clones, cDNA pool “Ribosomal”; 1A9, 1B2 and 4A4, cDNA pool “ATPase”).

^cResulted in enhanced tick feeding after mouse vaccination and tick challenge.

The sequence of hsp70 contained a 3’ untranslated region (UTR) of 299 bp before the poly-A tail. The clone 3E1 contained a cDNA identical to the beta-adaptin that produced a 27% inhibition of tick infestation and a 5% inhibition of molting to the nymphal stage after vaccination and tick challenge (Table 3). The complete sequence was determined for the clone 3E1 (Figure 1A), and contained an insert of 1,942 bp encoding for a predicted protein of 191 amino acids. The sequence of this protein was shorter than that for other beta-adaptins (Figure 1B), suggesting that it could encode for a beta-adaptin appendage or it may be a partial cDNA sequence because of a long 3’ UTR of 1,334 bp located before the poly-A tail.

The cDNA in clone 4G11 was identical to a chloride channel but it contained only a partial sequence (Figure 2A). This sequence protected against tick infestations and inhibited larval molting (Table 3). Chloride channels have been found in living organisms from bacteria to mammals, with some amino acid positions being conserved in all sequences (Figure 2A). As expected, phylogenetic analysis of chloride channel sequences demonstrated that the I. scapularis sequence comprised a sister group to other insect sequences that have been reported (Figure 2B).

Vaccination with ribosomal sequences had some inhibitory effect on tick infestations but did not affect molting (Table 3). The pool of ribosomal cDNAs included EST sequences coding for cellular and mitochondrial ribosomal proteins and translation factors (Table 4), and these genes are highly conserved across species. However, proteins encoded by I. scapularis ESTs were 43% to 95% identical to arachnida or insect sequences and 36% to 85% identical to mouse sequences (Table 4). The cDNA in clone 2C12 that was found to be identical to the beta-amyloid precursor protein (APP) contained a fragment encoding for the C-terminal of the protein (Figure 3), suggesting that it contains a partial cDNA with a long (1,400 bp) 3’ UTR. Nonetheless, the C-terminal sequence of the I. scapularis APP contained regions of amino acids identical to fly and mosquito sequences (Figure 3). Vaccination with this cDNA resulted in 8% enhancement of larval feeding (Table 3). Vaccination with cDNA clone 4F1 resulted in enhanced larval feeding (Table 3). The complete sequence of clone 4F1 cDNA was determined and contained an insert of 2,475 bp with 30 bp and 66 bp of 5’ and 3’ UTR, respectively and a poly-A tail of 114 bases.

A

cgATGCAGGCGATGACGGGCTTTGCGGTGCAGTTCAACAAAAACAGTTTCGGGCTGACTCCAGCTCAGCCGCTGCAGTTGCAGATTCCCCTGCAGCCCAACTTCCCAGCTGATGCGAGCTTGCAGCTGGGAACCAACGGTCCCGTGCAGAAGATGGACCCCCTCACCAACCTTCAGGTGGCCATCAAGAACAATGTGGACGTGTTCTACTTCAGCTGCCTGGTGCCCATGCACGTGCTGAGCACGGAGGACGGCCTGATGGACAAGCGGGTGTTCCTGGCCACCTGGAAAGACATCCCCGCCCAAAACGAGGTCCAGTACACCCTCGACAACGTCAACCTCACTGCAGACCAAGTTTCCCAGAAGCTGCAGAACAACAACATTTTCACGATAGCCAAGAGGAACGTGGACGGCCAGGACATGCTGTACCAGTCCCTGAAGCTCACCAACGGCATTTGGGTGTTGGCGGAGCTCAAGATACAGCCCGGCAATCCAAGGATCACGTTGTCTTTGAAGACAAGAGCACCTGAAGTGGCAGCAGGTGTACAACAAACTTACGAACTCATTCTACACAGCTGAggctgctgtgaatgaaactcttctcccacccccttcttttgatggcagtcaatgtctcgtttcattttcttgttttcttttgcggcgtgctacggaacaaggtcctacattcccaagttatatggtgttgtcgcgtagggggcagagtgccgctgagcccgcgacagccttgtttctgaggagagccgaacgcaccacttcgaaaaagaaaaagtgaaaacggaaaaatgaaaaattttccagttgcttcaaattaacattcctcgtagtcagtctgtggccgttgagtttggtgtaaagaagaaaaaggtgtctcttttagtgaaaatggttgctttttattggtatcccctatcacaccgagcacgaacataagaaatcctgacaaggattctcctttagttgtattatggtggctggagcacacgaggcacctgttgccaattcgacccagcaaatgcccaattctcaagatttgagttcattgaggttgttttgctcctccccccccaccccccaactttgtcgttggattgtctaacagtgtaaatgggcgacgactcgttattctttttttcttcattctttctttttgttgtcacgcgccccgggggacgcgacacaacttatgtgcataattgattttcacaggctgcgacgcagtctgtaaaagaaggggaagtgaaactctgctccgccgctgctagtgtcatcacgggacgaccatcgcgttttctctgactatttaaacaaaactgcatagcttagggggcagtctgtgcaaagtggaacaaccaaactgagccctgccctttcggtgtgtgtacaagcatctctgtgtaacatgaactactttacatgaactacattgcatgaacgggagaagtttagttgtttttttgttttttttttcaggtgactatgtcaacagattagaaccattttttggaacggctggaaagataaccgctcattttgtttctactaaaagactacgaaaagtgttgactttttgcatcggtttggcaacgtttgtttggcatgcatgtagttgagcgtaatggtatcacccctcgtaaacaataacagtgcaatggagcagtactgtagtgtccattaaagagcgagagtttggttaaaggttgttaattgaggtccgtgttatcctttgagtaggagagcggcactttttgcaaatagcgctgctgggggcgtcatatctgccctccaaaacatgcacattttaagtgtgaattgttgcggcggcttgtacaagtatgtgtgttatgtgtagaaaaagaactcttaattaaaatatttgtggccaaaacgtcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

B

M. musculus (747) LQHMTDFAIQFNKNSFGVIPSTPLAIHTPLMPNQSIDVSLPLNTLGPVMK

D. melanogaster (731) MQPMTNFAIQLNKNSFGLVPASPMQ-AAPLPPNQSIEVSMALGTNGPIQR

H. sapiens (68) LQHMTDFAIQFNKNSFGVIPSTPLAIHTPLMPNQSIDVSLPLNTLGPVMK

I. scapularis (1) MQAMTGFAVQFNKNSFGLTPAQPLQLQIPLQPNFPADASLQLGTNGPVQK

Consensus (748) LQHMTDFAIQFNKNSFGLIPATPLQIHTPLMPNQSIDVSLPLNTNGPVQK

M. musculus (797) MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMERQVFLATWKDIPN

D. melanogaster (780) MEPLNNLQVAVKNNIDIFYFACLVHGNVLFAEDGQLDKRVFLNTWKEIPA

H. sapiens (118) MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMERQVFLATWKDIPN

I. scapularis (51) MDPLTNLQVAIKNNVDVFYFSCLVPMHVLSTEDGLMDKRVFLATWKDIPA

Consensus (798) MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMDKRVFLATWKDIPN

M. musculus (847) ENELQFQIKECHLNADTVSSKLQNNNVYTIAKRNVEGQDMLYQSLKLTNG

D. melanogaster (830) ANELQYTLSGVIGTTDGIASKMTTNNIFTIAKRNVEGQDMLYQSLKLTNN

H. sapiens (168) ENELQFQIKECHLNADTVSSKLQNNNVYTIAKRNVEGQDMLYQSLKLTNG

I. scapularis (101) QNEVQYTLDNVNLTADQVSQKLQNNNIFTIAKRNVDGQDMLYQSLKLTNG

Consensus (848) ENELQFTIKEVHLTADTVSSKLQNNNIFTIAKRNVEGQDMLYQSLKLTNG

M. musculus (897) IWILAELRIQPGNPNYTLSLKCRAPEVSQYIYQVYDSILKN-

D. melanogaster (880) IWVLLELKLQPGNPEATLSLKSRSVEVANIIFAAYEAIIRSP

H. sapiens (218) IWILAELRIQPGNPNYTLSLKCRAPEVSQYIYQVYDSILKN-

I. scapularis (151) IWVLAELKIQPGNPRITLSLKTRAPEVAAGVQQTYELILHS-

Consensus (898) IWILAELKIQPGNPNYTLSLKCRAPEVAQYIYQVYDSILKS

Figure 1. Analysis of clone 3E1 identical to beta-adaptin. (A) Nucleotide sequence of complete cDNA. Non-coding sequence is shown in lower case letters and coding sequence is shown in capital letters with translation initiation and termination codons in bold letters. (B) Alignment of M. musculus (GenBank accession number XP_109938), D. melanogaster (CAA53509) and Homo sapiens (AAA35583) protein sequences and the translation product of clone 3E1 identified as I. scapularis beta-adaptin appendage (AY296113). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 3 of 4 sequences are shown in blue.

A

E. coli (4) DTPSLETPQAARLRRRQLIRQLLERDKTPLAILFMAAVVGTLVGLAA-VA

O. mossambicus (98) DLKEGVCLSALWFNH--------EQ----------CCWTSNETTFAERDK

X. laevis (146) DLKEGICLPWFWFNH--------EQ----------CCWQSNNVTFEDRNN

I. scapularis (1) DLKEGICPQAFWLNK--------EQ----------CCWASNDTFFKG-DD

C. elegans (141) DLKTGVCADRFWLDH--------EH----------CCWSSNDTFYKD-DD

D. melanogaster (223) DLKHGICPPAFWFNR--------EQ----------CCYPAKQSVFEE-GN

L. major (114) AFRSGICANFFWLGR-------------------------N-MCCVDCRE

A. gambiae (272) DLKFGICPQAFWLNR--------EQ----------CCWSSNETSFDS-GN

M. musculus (155) DLKEGICLSALWYNH--------EQ----------CCWGSNETTFEERDK

S. tuberosum (108) GFKLLLTSNLMLDGK-----------------------------------

S. cerevisiae (102) NWKTGHCQRNWLLNKS-------------------FCCNGVVNEVTSTSN

Consensus (272) DLK GIC AFWLNR EQ CCW SN T F D

E. coli (53) FDKGVAWLQNQRMGALVHTADNYPLLLTVAFLCSAVLAMFGYFLVRKYAP

O. mossambicus (130) CPQWKSWAELILGQ--AEGPGSYIMNYFMYIYWALSFAFLAVCLVKVFAP

X. laevis (178) CPEWRSWSQLVLGR--SEGAFPYILNYFMYVMWALLFSLLAVLLVRNFAP

I. scapularis (32) CKQWYRWPEMFDSGMDKDGAGFYLLSYLLYVMWSVLFATLAVMLVRTFAP

C. elegans (172) CKAWTKWPWMLNYYN-SSSFLFLFLEWIFYIGWAVAMSTLAVLFVKIFAP

D. melanogaster (254) CSTWKTWPEIFGLD--RNGTGPYIVAYIWYVLWALLFASLSASLVRMFAP

L. major (138) CGEYYSWGEFFLGR---DNHVVAFVDFVMYVSFSTMAAVTAAYLCKTYAP

A. gambiae (303) CSQWYAWSEIFTSS--REGFGAYVISYFFYIMWAMLFALLAASLVRMFAP

M. musculus (187) CPQWKTWAELIIGQ--AEGPGSYIMNYIMYIFWALSFAFLAVSLVKVFAP

S. tuberosum (123) ----------------------YFQAFAAFAGCNVFFATCAAALCAFIAP

S. cerevisiae (133) LLLKRQEFECEAQG-LWIAWKGHVSPFIIFMLLSVLFALISTLLVKYVAP

Consensus (322) C W W EL EG YIL YIMYILWALLFA LA LVK FAP

E. coli (103) EAGGSGIPEIEGALE---DQRPVRWWRVLPVKFFGGLGTLGGGMVLGREG

O. mossambicus (178) YACGSGIPEIKTILSGF-IIRGYLGKWTLMIKTITLVLAVASGLSLGKEG

X. laevis (226) YACGSGIPEIKTILSGF-IIRGYLGKWTLIIKTMTLVLAVSSGLSLGKEG

I. scapularis (82) YACGSGIPEIKTILSGF-IIRGYLGKWTLTIKSVCLVLAVGAGLSLGKEG

C. elegans (221) YACGSGIPEIKCILSGF-VIRGYLGKWTFIIKSVGLILSSASGLSLGKEG

D. melanogaster (302) YACGSGIPEIKTILSGF-IIRGYLGKWTLLIKSVGLMLSVSAGLTLGKEG

L. major (185) YASGGGIAEVKTIVSGH-HVKRYLGGWTLITKVVGMCFSTGSGLTVGKEG

A. gambiae (351) YACGSGIPEIKTILSGF-IIRSYLGKWTLIIKSVGIMLSVSAGLSLGKEG

M. musculus (235) YACGSGIPEIKTILSGF-IIRGYLGKWTLMIKTITLVLAVASGLSLGKEG

S. tuberosum (151) AAAGSGIPEVKAYLNG-IDAHSILAPSTLLVKIFGSILGVSAGFVVGKEG

S. cerevisiae (182) MATGSGISEIKVWVSGFEYNKEFLGLLTLVIKSVALPLAISSGLSVGKEG

Consensus (372) YACGSGIPEIKTILSGF IIRGYLGKWTLIIKSVGLVLAVSSGLSLGKEG

E. coli (150) PTVQIGGNIGRMV----------LDIFRLKG--DEARHTLLATGAAAGLA

O. mossambicus (227) PLVHVACCCGNIF----------SYLFPKYSKNEAKKREVLSAASAAGVS

X. laevis (275) PLIHVACCCGNIL----------CHLFTKYRKNEAKRREVLSAAAAAGVS

I. scapularis (131) PLVHVACCIGNIF----------SYLFPKYGKNEAKKREILSAAAAAGVS

C. elegans (270) PMVHLACCIGNIF----------SYLFPKYGLNEAKKREILSASAAAGVS

D. melanogaster (351) PMVHIASCIGNIF----------SHVFPKYGRNEAKKREILSAAAAAGVS

L. major (234) PFVHIGACVGGII----------SGALPSYQQ-EAKERELITAGAGGGMA

A. gambiae (400) PMVHIASCIGNIL----------SYLFPKYGRNEAKKREILSAAAAAGVS

M. musculus (284) PLVHVACCCGNIF----------SYLFPKYSTNEAKKREVLSAASAAGVS

S. tuberosum (200) PMVHTGACIANLLGQGGSRKYHLTWKWLKYFKNDRDRRDLITCGAAAGVA

S. cerevisiae (232) PSVHYATCCGYLL----------TKWLLRDTLTYSTQYEYLTAASGAGVA

Consensus (422) PLVHIA CIGNIL SYLFPKY KNEAKKREILSAAAAAGVS

E. coli (188) AAFNAPLAGILFIIEEMRPQ--FRYTLISIKAVFIGVIMSTIMYRIFNHE

O. mossambicus (267) VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFVLRSINPFGN

X. laevis (315) VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFTLRSINPFGN

I. scapularis (171) VAFGAPIGGVLFSLEEVSYY--XPLKTLWRSFFCALVAASVLRSINPFGN

C. elegans (310) VAFGAPIGGVLFSLEEASYY--FPLKTMWRSFFCALVAGIILRFVNPFGS

D. melanogaster (391) VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFCALIAAFVLRSLTPFGN

L. major (273) VAFGAPVGGVIFALEDVSTS--YNFKALMAALICGVTAVLLQSRVDLWHT

A. gambiae (440) VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFCALIAAFILRSINPFGN

M. musculus (324) VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFVLRSINPFGN

S. tuberosum (250) AAFRAPVGGVLFALEEIASW--WRSALLWRTFFTTAIVAMVLRSLIQFCR

S. cerevisiae (272) VAFGAPIGGVLFGLEEIASANRFNSSTLWKSYYVALVAITTLKYIDPFRN

Consensus (472) VAFGAPIGGVLFSLEEVSYY FPLKTLWRSFF ALVAA VLRSINPFGN

E. coli (236) VA----------LIDVGKLSDAPL

O. mossambicus (315) SR----------LVLFYVEYHTPW

X. laevis (363) SR----------LVLFYVEFHAPW

I. scapularis (219) DH----------LVMFYVEYDFPW

C. elegans (358) NQ----------TSLFHVDYMMKW

D. melanogaster (439) EH----------SVLFFVEYNKPW

L. major (321) GR----------IVQFSVNYQHNW

A. gambiae (488) EH----------SVLFYVEYNKPW

M. musculus (372) SR----------LVLFYVEYHTPW

S. tuberosum (298) GGNCGLFGQGGLIMFDVNSGVSNY

S. cerevisiae (322) GR----------VILFNVTYDRDW

Consensus (522) LVLFYVEY PW

B

Figure 2. Analysis of clone 4G11 identical to chloride channel. (A) Alignment of M. musculus (XP_134186), D. melanogaster (AAM76180), Solanum tuberosum (T07608), Oreochromis mossambicus (AAD56388), A. gambiae (EAA11899), C. elegans (NP_495940), Leishmania major (strain Friedlin) (T02805), Saccharomyces cerevisiae (P37020), Escherichia coli K12 (AAC73266), and Xenopus laevis (CAA71071) protein sequences and the translation product of clone 4G11 identified as a fragment of I. scapularis chloride channel (AY296114). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 6-10 of 11 sequences are shown in blue. (B) Phylogenetic tree constructed from analysis of chloride channel protein sequences based on a sequence distance method utilizing the Neighbor Joining algorithm of Saitou and Nei (1987).

D. melanogaster PHAQGFIEVDQNVTTHHPIVREEKIVPNMQINGYENPTYKYFE

I. scapularis PQAQGFVQVDQGALPASPEER---HLASMQVNGYENPTYKYFE

A. gambiae PHAQGFVEVDQAVGAPVTPEE--RHVANMQINGYENPTYKYFE

Consensus PHAQGFVEVDQ V P ER HVANMQINGYENPTYKYFE

Figure 3. Analysis of clone 2C12 identical to beta-amyloid precursor protein. Alignment of D. melanogaster (AF181628) and A. gambiae (EAA07868) protein sequences and the translation product of clone 2C12 identified as I. scapularis beta-amyloid peptide (ß-AP) (AY296115). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 2 of 3 sequences are shown in blue.

Table 4. Characterization of I. scapularis ESTs encoding for ribosomal proteins

EST clone	Predicted protein	Identical amino acids	Species	GenBank accession number
4F7 1A2	Elongation factor 1-alpha	95% 85%	Neacarus texanus Mus musculus	AAK12660 NP_031932
1A10	Elongation factor-2	88% 80%	Mastigoproctus giganteus Mus musculus	AAK12348 BAC26203
1C11	eIF-5A	65% 59%	Drosophila melanogaster Mus musculus	AAM68297 XP_203336
1F6 2C3	RpS4	79% 75%	Spodoptera frugiperda Mus musculus	AAL26580 AAH09100
2B8	RpS11	92% 80%	Dermacentor variabilis Mus musculus	AAO92287 XP_133477
2F8	Laminin receptor 1 (RpSA)	66% 73%	Anopheles gambiae Mus musculus	EAA00413 NP_035159
2F10	RpL3	70% 68%	Spodoptera frugiperda Mus musculus	AAL62468 AAH09655
3A10	RpL7A	55% 60%	Drosophila melanogaster Mus musculus	NP_511063 A30241
3D10	Ribophorin I	57% 50%	Drosophila melanogaster Mus musculus	AAN71150 BAC26679
3G9	RpS8	70% 71%	Spodoptera frugiperda Mus musculus	AAL62472 XP_134904
3G10	RpL27A	42% 36%	Spodoptera frugiperda Mus musculus	AAK92158 XP_137118
4D11	Proteasome subunit	60% 55%	Drosophila melanogaster Mus musculus	NP_524115 NP_035315
4D12	Proteasome/Signalosome subunit	43% 56%	Anopheles gambiae Mus musculus	EAA11895 AAC33900
4E7	Proteasome subunit	84% 85%	Anopheles gambiae Mus musculus	EAA10351 NP_036096

The sequences of I. scapularis ESTs identical to ribosomal proteins pooled for DNA vaccination as described in Almazán et al. (2003), were compared to all non-redundant sequences in GenBank DNA and protein databases (1,419,727 sequences total; Apr-09-2003) using BLASTX 2.2.6 (Altschul et al., 1997). The percent of identical amino acids to arachnida or insect and mouse sequences are shown together with their corresponding GenBank accession number. The GenBank accession numbers for I. scapualris sequences are shown on Table 1.

1 50

M. musculus (1) ------------------------MAGACGKPHMSPASLPGKRRLEPDQE

D. melanogaster (1) MTKKLALKRRGKDSEPTNEVVASSEASENEEEEEDLLQAVKDPGEDSTDD

H. sapiens (1) ----------------------------SVRPEKRRSEPELEPEPEPEPP

A. gambiae (1) ---------------------QENLLGSIENEGEDSSDSDGEYATDDDED

I. scapularis (1) ----------------------MGPKTLSKQPAKASSSTSKRTAGPTISK

Consensus (1) P S E A D D D

51 100

M. musculus (27) LQIQEPPLLSD-PDSSLSDSEESVFSGLEDSGSDSSEEDTEGVA----GS

D. melanogaster (51) EGIDQEYHSDSSEELQFESDEEGNYLGRKQSSSAEEDEESSDEEDN---E

H. sapiens (23) LLCTSPLSHSTGSDSGVSDSEESVFSGLEDSGSDSSEDDDEGDEEGEDGA

A. gambiae (30) DVLSFESLNSDGEE---EDEEEDAGTTLEEVEREAEEDDDEEDAERKQRE

I. scapularis (29) QTEDSDDEGSSSAYSDLEDSEGADSSDSNDLSDTEASEDDYDDSQDEENT

Consensus (51) I E SS DS LEDSEES FSGLEDS SDSSEEDDEDDAE

101 150

M. musculus (72) SGDEDNHRAEETSEELAQAAPLCSRTEE--------------AGALAQDE

D. melanogaster (98) EEESTDGEEVEDEEKDSKSKQTDDKPSGSGAASKKALTAELPKRDSSKPE

H. sapiens (73) LDDEGHSGIKKTTEEQVQASTPCPRTEM--------------ASARIGDE

A. gambiae (77) EQFESDDEPLPDDLKLGRIEDVLGTGEKKTRGLGVFPPVPKRKGKAAQDE

I. scapularis (79) KITLTGVEGKDLELRGKDQEAPVESGKRSAWHRQQEDAKEDRRTQVVEDE

Consensus (101) DET E E EEK A R E K A DE

151 200

M. musculus (108) YEE-DSSDEEDIRNTVGNVPLAWYDEFPHVGYDLDGKRIYKPLRTRDELD

D. melanogaster (148) YQDSDTSDEEDIRNTVGNIPMHWYDEYKHIGYDWDAKKIIKPPQG-DQID

H. sapiens (109) YAE-DSSDEEDIRNTVGNVPLEWYDDFPHVGYDLDGRRIYKPLRTRDELD

A. gambiae (127) YAAGDTSDEEDIRNTVGNIPMHWYDEYKHVGYDWDAKKIIKAKKG-DAID

I. scapularis (129) YAF-DSSDEEDVRNTVGNIPLEWYEHYPHIGYDLEGKPILKPPRV-SDLD

Consensus (151) YAE DSSDEEDIRNTVGNIPL WYDEYPHVGYDLDGKKIIKP R DELD

201 250

M. musculus (157) QFLDKMDDPDFWRTVQDKMTGRDLRLTDEQVALVHRLQRGQFGDSGFNPY

D. melanogaster (197) EFLRKIEDPDFWRTVKDPLTGQDVRLTDEDIALIKRIVSGRIPNKDHEEY

H. sapiens (158) QFLDKMDDPDYWRTVQDPMTGRDLRLTDEQVALVRRLQSGQFGDVGFNPY

A. gambiae (176) DFLQRMEDPNFWRTVTDPQTGQKVVLSDEDIGLIKRIMSGRNPDAEYDDY

I. scapularis (177) DFLRKMDDPNYWRTVKDKSTGQDVVLTDEDVDLIQRLQKGQFPSSTTDPY

Consensus (201) DFL KMDDPDFWRTV DPMTGQDVRLTDEDVALIKRLQSGQFPDS FDPY

251 300

M. musculus (207) EPAVDFFSGDIMIHPVTNRPADKRSFIPSLVEKEKVSRMVHAIKMGWIKP

D. melanogaster (247) EPWIEWFTSEVEKMPIKNVPDHKRSFLPSVSEKKRVSRMVHALKMGWMKT

H. sapiens (208) EPAVDFFSGDVMIHPVTNRPADKRSFIPSLVEKEKVSRMVHAIKMGWIQP

A. gambiae (226) EPFIEWFTSEVEKMPIRNIPESKRSFLPSKAEKHKIGRYVHALKMGWMKT

I. scapularis (227) EPFEDIFSHETMIHPVTRHPPQKRSFVPSRIEKAMVSKMVHAIKMGWIKP

Consensus (251) EPFIDFFS EVMIHPVTN P KRSFIPSLVEK KVSRMVHAIKMGWIKP

301 350

M. musculus (257) RRPHD------PTPSFYDLWAQEDPNAVLG-RHKMHVPAPKLALPGHAES

D. melanogaster (297) TEEVEREKQAKRGPKFYMLWETDTSREHMR-RIHDPVSAPKRDLPGHAES

H. sapiens (258) RRPRD------PTPSFYDLWAQEDPNAVLG-RHKMHVPAPKLALPGHAES

A. gambiae (276) MAEKRRLEAIRRQPKFYMLWTTDHGKEEMR-RIHDHVAAPKRMLPGHAES

I. scapularis (277) RVKKH------DPERFSLLWDKDDSTAGSNERMQRHIPAPKMKLPGHEES

Consensus (301) R KD PKFYMLW DD A L RI HVPAPKL LPGHAES

351 400

M. musculus (300) YNPPPEYLPTEEERSAW--MQQEPVERKLNFLPQKFPSLRTVPAYSRFIQ

D. melanogaster (346) YNPPPEYLFDAKETKEWLKLKDEPHKRKLHFMPQKFKSLREVPAYSRYLR

H. sapiens (301) YNPPPEYLLSEEERLAW--EQQEPGERKLSFLPRKFPSLRAVPAYGRFIQ

A. gambiae (325) YNPPPEYLFDEKELEEWNKLANQPWKRKRAYVPQKYNSLREVPGYTRYVK

I. scapularis (321) YNPPAEYLFTEEEEAKWR--EQEPEERRINFLPAKYPCLRAVPAYERFIE

Consensus (351) YNPPPEYLFTEEE W L QEP ERKL FLPQKFPSLR VPAYSRFI

401 450

M. musculus (348) ERFERCLDLYLCPRQRKMRVNVDPEDLIPKLPRPRDLQPFPVCQALVYRG

D. melanogaster (396) ERFLRCLDLYLCPRAKRVKLNIDAEYLIPKLPSPRDLQPFPTVESMVYRG

H. sapiens (349) ERFERCLDLYLCPRQRKMRVNVDPEDLIPKLPRPRDLQPFPTCQALVYRG

A. gambiae (375) ERFLRCLDLYLAPRMRRSRVAVGAEYLIPKLPSPRDLQPFPTLQNLIYTG

I. scapularis (369) ERFERCLDLYLCPRQRKMRVNVDAEDLIPQLPKPKDLQPFPSIQSIVYEG

Consensus (401) ERFERCLDLYLCPRQRKMRVNVDAEDLIPKLPRPRDLQPFPTIQALVYRG

451 500

M. musculus (398) HSDLVRCLSVSPGGQWLASGSDDGTLKLWEVATARCMKTVHVGGVVRSIA

D. melanogaster (446) HTDLVRSVSVEPKGEYLVSGSDDKTVKIWEIATGRCIRTIETDEVVRCVA

H. sapiens (399) HSDLVRCLSVSPGGQWLVSGSDDGSLRLWEVATARCVRTVPVGGVVKSVA

A. gambiae (425) HTSLIRCISVEPKGEYIVTGSDDMTVKIWEISTARCIRTIPTGDIVRSVA

I. scapularis (419) HTDCVLCLSLEPAGQFFASXSEDGTVRIWELLTGXCLKKFQFEAPVKSVA

Consensus (451) HTDLVRCLSVEPGGQWLVSGSDDGTVKIWEIATARCIRTI GGVVRSVA

501 550

M. musculus (448) WNPNPTICLVAAAMDDAVLLLNPALGDRLLVGSTDQLLEAF----TPPEE

D. melanogaster (496) WCPNPKLSIIAVATGNRLLLVNPKVGDKVLVKKTDDLLAEAPSQDVIESE

H. sapiens (449) WNPSPAVCLVAAAVEDSVLLLNPALGDRLVAGSTDQLLSAF----VPPEE

A. gambiae (475) WCPNSKISLVAAASGKRVLLINPKVGDYMLVKKTDDLLTEAPRSDTVDSE

I. scapularis (469) WCP--VVVPMKLCVDKTVSMLDAGVTDKLLPFTTGHRVVCPPRRVLGPGG

Consensus (501) WCPNP I LVAAAVD VLLLNPAVGDKLLV STD LL P V P E

551 600

M. musculus (494) PALQPARWLEVSEEEHQRGLRLRICHSKPVTQVTWHGRGDYLAVVLSSQE

D. melanogaster (546) RIKTAVQWSNAEADEQEKGVRVVITHFKPIRQVTWHGRGDYLATVMPEGA

H. sapiens (495) PPLQPARWL