**3. Strategy for cloning of vulture TLR1 and IκBα**

In order to identify key components of the vulture system for sensing of pathogens, we screened a cDNA library from vulture peripheral blood monuclear cells (PBMC) using spe‐ cific probes for TLR1 and IκBα.

Since the majority of toll-like receptors are expressed in leukocytes and lymphoid tissues in human and other vertebrates, we decided to use vulture PBMC as the source of RNA to ob‐ tain a specific probes for TLR1 and IκBα and to construct a cDNA library. Using this strat‐ egy we cloned cDNAs encoding for griffon vulture (*Gyps fulvus*) orthologues of mammalian

TLR1 (CD281) and for the alpha inhibitor of NF-κB (IκBα). The tissue and cell expression pattern of vulture TLR1 and IκBα were analyzed by real-time RT-PCR and correlated with the ability to respond to various pathogenic challenges.

(Amersham, Barcelona, Spain) denatured in 1.5 M NaCl/0.5M NaOH, neutralised in 1.5 M NaCl/0.5 Tris (pH 8.0) and fixed using a cross-linker oven (Stratagene). The filters were then pre-incubated with hybridisation buffer (5XSSC [1XSSC is 150 mM NaCl, 15 mM trisodium citrate, pH 7.7], 0.1% N-laurylsarcosine, 0.02% SDS and 1% blocking reagent (Roche)) at 65 °C for 1 h and then hybridised with hybridisation buffer containing the DIG-labelled probe, overnight at 65 °C. The membranes were washed at high stringency (2XSSC, 0.1% SDS; 2x5 min at ambient temperature followed by 0.5XSSC, 0.1% SDS; 2x15 min at 65 °C). DiG-label‐ led probes were detected using phosphatase-labelled anti-digoxygenin antibodies (Roche) according to the manufacturer's instructions. Positive plaques on membranes were identi‐ fied, isolated in agar plugs, eluted in 1 ml of SM buffer (0.1M NaCl, 10 mM MgSO4, 0.01% gelatin, 50 mM Tris-Hcl, pH 7.5) for 24 h at 4°C and replated. The above screening protocol was then repeated. Individual positive plaques from the secondary screening were isolated in agar plugs and eluted in SM buffer. The cDNA inserts were recovered using the Exassist/ SOLR system (Stratagene). Individual bacterial colonies containing phagemid were grown up in LB broth (1% NaCl, 1% trytone, 0.5% yeast extract, pH 7.0) containing 50 μg/ml ampi‐ cillin. Phagemid DNA was purified using a Bio-Rad plasmid mini-prep kit and sequenced.

Identification of Key Molecules Involved in the Protection of Vultures Against Pathogens and Toxins

http://dx.doi.org/10.5772/54191

247

**4. Structural analysis of vulture TLR1 and IκBα sequences**

was made using the webtool SUMOplot™.

DQ480086 and EU161944, respectively.

ysis of TLR1 transcripts in vulture cells.

**4.1. Vulture TLR1**

Sequences were analyzed using the analysis software LaserGene (DNAstar, London, UK) and the analysis tools provided at the expasy web site (http://www.expasy.org). PEST regions are sequences rich in Pro, Glu, Asp, Ser and Thr, which have been pro‐ posed to constitute protein instability determinants. The analysis of the PEST region for the putative protein was made using the webtool PESTfind at http:// www.at.embnet.org/toolbox/pestfind. The potential phosphorylation sites were calculat‐ ed using the NetPhos 2.0 prediction server at http://www.cbs.dtu.dk/services/NetPhos. The prediction of the potential attachment of small ubiquitin-related modifier (SUMO)

The alignment of vulture TIR domain sequences with TLR-1 from other species and of the vulture IκBα sequences with IκBα from other species was done using the program ClustalW v1.83 with Blosum62 as the scoring matrix and gap opening penalty of 1.53. Griffon vulture TLR-1 and IκBα sequences were deposited in the Genbank under accession numbers

The screening of the vulture PBMC cDNA library for TLR1 yielded seven clones with identi‐ cal open reading frame (ORF) sequences. The fact that the screening of 500,000 vulture cDNA clones resulted in 7 identical sequences suggested that this TLR receptor is broadly represented in PBMC, possibly illustrating its important role in pathogen recognition during vulture innate immune response. This result was consistent with the real time RT-PCR anal‐

### **3.1. Design of specific probes for vulture TLR1 and IκBα**

To obtain specific probes for vulture TLR1 and IκBα, total RNA was isolated from vulture PBMC and from cells and tissues using the Ultraspec isolation reagent (Biotecx Laboratories, Houston TX, USA). Ten micrograms of total RNA was heated at 65 °C for 5 min, quenched on ice for 5 min and subjected to first strand cDNA synthesis. The RNA was reverse tran‐ scribed using an oligo dT12 primer by incubation with 200 U RNase H- reverse transcriptase (Invitrogen, Barcelona, Spain) at 25°C for 10 min, then at 42°C for 90 min in the presence of 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 30 U RNase-inhibitor and 1mM dNTPs, in a total volume of 20 μl.

For the vulture TLR probe, a partial fragment of 567 bp showing sequence similarity to hu‐ man TLR-1 was amplified by PCR from vulture PBMC cDNA using two oligonucleotide pri‐ mers TLR1/2Fw (5'-GAT TTC TTC CAG AGC TG–3') and TLR1/3Rv (5'-CAA AGA TGG ACT TGT AAC TCT TCT CAA TG -3'), which were designed based on regions of high ho‐ mology among the sequences of human and mouse TLR1 (GenBank, accession numbers NM\_003263 and NM\_030682, respectively). Cycling conditions were 94°C for 30 s, 52°C for 30 s and 72°C for 1.5 min, for 30 cycles.

For the vulture IκBα probe, a partial fragment of 336 bp showing sequence similarity to hu‐ man and chicken IκBα was amplified by PCR from vulture PBMC cDNA using two oligonu‐ cleotide primers IκBα-Fw (5'-CCT GAA CTT CCA GAA CAA C-3') and IκBα-Rv (5'-GAT GTA AAT GCT CAG GAG CCA TG-3'), which were designed based on regions of high ho‐ mology among the sequences of human and chicken IκBα (GenBank, accession numbers M69043 and S55765, respectively). Cycling conditions were 94°C for 30 s, 52°C for 30 s and 72°C for 1.5 min, for 30 cycles.

The obtained PCR products were cloned into pGEM-T easy vector using a TA cloning kit (Promega, Barcelona, Spain) and sequenced bidirectionally to confirm their respective spe‐ cificities. These fragments were DIG-labelled following the recommendation of the manu‐ facturer (Roche, Barcelona, Spain) and used as probes to screen 500 000 plaque colonies of the vulture-PBMC cDNA library.

### **3.2. cDNA library construction and screening**

Total RNA (500 μg) was extracted from PBMC (pooled from 6 birds) using the Ultraspec iso‐ lation reagent (Biotecx). mRNA (20 μg) was extracted by Dynabeads (Dynal biotech-Invitro‐ gen, Barcelona, Spain) and used in the construction of a cDNA library in Lambda ZAP vector (Stratagene, La Jolla, CA, USA) by directional cloning into EcoRI and XhoI sites. The cDNA library was plated by standard protocols at 50 000 plaque forming units (pfu) per plate and grown on a lawn of XL1-Blue E. coli for 6-8 h. Screening of the library was per‐ formed with DIG labelled probes. Plaques were transferred onto Hybond-N+ membranes (Amersham, Barcelona, Spain) denatured in 1.5 M NaCl/0.5M NaOH, neutralised in 1.5 M NaCl/0.5 Tris (pH 8.0) and fixed using a cross-linker oven (Stratagene). The filters were then pre-incubated with hybridisation buffer (5XSSC [1XSSC is 150 mM NaCl, 15 mM trisodium citrate, pH 7.7], 0.1% N-laurylsarcosine, 0.02% SDS and 1% blocking reagent (Roche)) at 65 °C for 1 h and then hybridised with hybridisation buffer containing the DIG-labelled probe, overnight at 65 °C. The membranes were washed at high stringency (2XSSC, 0.1% SDS; 2x5 min at ambient temperature followed by 0.5XSSC, 0.1% SDS; 2x15 min at 65 °C). DiG-label‐ led probes were detected using phosphatase-labelled anti-digoxygenin antibodies (Roche) according to the manufacturer's instructions. Positive plaques on membranes were identi‐ fied, isolated in agar plugs, eluted in 1 ml of SM buffer (0.1M NaCl, 10 mM MgSO4, 0.01% gelatin, 50 mM Tris-Hcl, pH 7.5) for 24 h at 4°C and replated. The above screening protocol was then repeated. Individual positive plaques from the secondary screening were isolated in agar plugs and eluted in SM buffer. The cDNA inserts were recovered using the Exassist/ SOLR system (Stratagene). Individual bacterial colonies containing phagemid were grown up in LB broth (1% NaCl, 1% trytone, 0.5% yeast extract, pH 7.0) containing 50 μg/ml ampi‐ cillin. Phagemid DNA was purified using a Bio-Rad plasmid mini-prep kit and sequenced.
