Chapter 3 Materials and Methods

Chapter 3: Materials and Methods

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Chapter 3: Materials and Methods

3.1 Computational Methods

3.1.1 Public Databases

I searched several public biological databases in attempts to detect homologues of PRNP and their genomic context: Ensembl (http://www.ensembl.org/); National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/); DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/); The Institute for Genomic Research (TIGR; http://www.tigr.org/), Sanger Institute (http://www.sanger.ac.uk/) and local BLAST server for Sanger Institute zebrafish database on http://danio.mgh.harvard.edu/blast/blast_grp.html); Functional Annotation of Mouse (FANTOM; http://www.gsc.riken.go.jp/e/FANTOM/); The Fugu Genomics group at the Rosalind Franklin Centre for Genomics Research (http://Fugu.hgmp.mrc.ac.uk/); A Database of the Drosophila Genome (FlyBase; http://www.flybase. org/); Genoscope (http://www.genoscope.cns.fr/); Medaka Genome Database (M Base; http://mbase.bioweb.ne.jp/~dclust/medaka_top.html); and The Biology and Genome of C. elegans (WormBase; http://www.wormbase.org/).

3.1.2 Analysis of Nucleic Acids Sequences

3.1.2.1 Basic Analysis and Handling of Sequences

The Lasergene (DNASTAR, Madison WI, USA) and Vector NTI (InforMax, Frederick MD, USA) software packages were used for basic handling, storage and analyses of the nucleotide sequences. I used these programs to align cDNAs with genomic sequences (local alignment) and define gene features. I also used these software packages to align homologous sequences (global alignment) and to determine basic gene features such as length of exons/introns and their nucleotide context.

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Exon-intron structures of SPRN genes were determined using the Local Pair Alignment tool (Huang and Miller, 1991) on the ANGIS interactive web interface Biomanager (http://www.angis.org.au).

3.1.2.2 Design of PCR Primers

I used the MacVector 7.0 program (Oxford Molecular Group 2000) to design primers for the PCR experiments. As stringent as possible criteria for primer design were adjusted individually for each primer.

3.1.2.3 Analysis of Transposable Element Content

I used the slow speed option of the RepeatMasker program as a free web service (http://ftp.genome.Washington.edu/RM/RepeatMasker.html) to determine the relative content of interspersed repeats, small RNA, satellites, simple repeats and low complexity sequence in my nucleotide sequences.

3.1.2.4 Prediction of CpG Islands

CpG islands in the human, mouse, and rat genomic sequences were detected by using the EMBOSS free open source software package (Rice et al. 2000) run locally under the Linux server. The cpgplot program (Larsen et al. 1992) identifies a CpG island as a sequence region where over an average of 10 windows, GC percentage is over 50% and Obs/Exp ratio is over 0.6 in a minimum of over 100 bp.

3.1.2.5 Analysis of Genomic Sequences using NIX Interactive Tool

I used routinely the NIX interactive web tool (Williams et al. 1998; http://www.hgmp.mrc.ac.uk/NIX/) to annotate genomic sequences. This service enables analysis and annotation of genomic sequences using several programs simultaneously. The analysis includes the CpG island, promoter, poly-A signal site and exon

105 Chapter 3 Materials and Methods predictions, analysis of transposable element content, filtering of vector sequences, and BLASTN and BLASTP searches (Figure 3.1).

3.1.3 Analysis of Protein Sequences

3.1.3.1 Translation of Protein Sequences in silico

I used the Lasergene (DNASTAR, Madison WI, USA) and Vector NTI (InforMax, Frederick MD, USA) program packages to translate protein amino acid sequences from nucleic acid nucleotide sequences and also for basic handling, storage and analyses of the protein sequences.

3.1.3.2 Alignment of Protein Sequences

I aligned protein sequences using the program Cameleon v3.14 (Oxford Molecular 1995, now owned by Accelrys) implementing the algorithm of Taylor (1990). Multiple sequence alignments were edited manually. Final alignment figures were prepared using the CHROMA alignment editor (Goodstadt and Ponting, 2001) available free on the web (http://www.lg.ndirect.co.uk/chroma/index.htm).

3.1.3.3 Prediction of Signal Peptides and GPI-anchor Addition Sites

I used the SignalP program to predict signal peptide cleavage sites (Nielsen et al., 1997) as free web service (http://www.cbs.dtu.dk/services/SignalP/). GPI-anchor addition sites were predicted using the bigPI-predictor (Eisenhaber et al., 1999) also as free web service (http://mendel.imp.univie.ac.at/sat/gpi/gpi_server.html).

3.1.3.4 Computational Prediction of Tammar Wallaby PrP Structure

The homology model of tammar wallaby PrP structure was built by submitting the primary protein amino acid sequence to the SWISS-MODEL, automated comparative modelling server available as free web service (http://swissmodel.expasy.org/).

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Figure 3.1 An example of the NIX output. The NIX analysis enables annotation of genomic sequences using a number of programs simultaneously. CpG island prediction: GRAIL/cpg. Promoter prediction: GRAIL/polIIprom, TSSW/Promotor, GENSCAN/Prom, FGenes/Prom. Exon prediction: Fex, Hexon, MZEF, Genemark, GRAIL/exons, GRAIL/gap2, Genefinder, Fgene, GENSCAN, FGenes. BLASTP searches: BLAST/trembl, BLAST/swissprot. BLASTN searches: BLAST/est, BLAST/embl-, BLAST/gss, BLAST/sts. Poly-A site prediction: Polyah, GENSCAN/polya, FGenes/polya, GRAIL/polya. Vector and contamination check: BLAST/vector, BLAST/ecoli. Transposable element content: RepeatMasker. tRNAs: tRNAscan-SE. Sequence, nucleotide sequence in bp. This figure was downloaded from http://www.hgmp.mrc.ac.uk/NIX/.

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I visualized and analysed protein structures by using the program Swiss-PdbViewer (http://au.expasy.org/spdbv/) run under MacOSIX. I aligned the model of tammar wallaby PrP structure with the experimental mouse (1AG2) and bovine (1DWY) PrP structures deposited in the Protein Data Bank (http://www.rcsb.org/pdb/index.html/).

3.1.3.5 Analysis of Evolutionary Distances

I calculated evolutionary distances in a set of aligned PrPs using the free MEGA2 program (Kumar et al., 2001; http://www.megasoftware.net) run locally under Windows. Evolutionary distances were defined as a number of amino acid substitutions between a pair of sequences. Using the Complete-Delete option to remove alignment gaps, I determined number of valid common sites and number of sites different between two sequences. I then calculated percent of identity between a pair of sequences manually.

3.1.3.6 Protein Amino Acid Pattern Search

I used the ScanProsite program available as free web service (http://au.expasy.org/tools/scanprosite/) to search the SWISS-PROT database with the defined marsupial PrP pattern [T-T-T-T-T-T-K].

3.1.4 Cross-Species Comparisons

3.1.4.1 Global Alignments of Long Genomic sequences

I used the VISTA free web server (Mayor et al. 2000; http://www-gsd.lbl.gov/VISTA/) to produce global alignments of long genomic sequences.

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3.1.4.1.1 Alignment of PRNP Genomic Context between Mammals and Fish

I aligned human (chr20: 4558866-4938939 bp; Ensembl human v12.31.1), mouse (chr2: 132862228-133103751 bp; Ensembl mouse v12.3.1), rat (chr3: 112821949-113040637 bp; Ensembl rat v11.2.1), Fugu (chr_scaffold_155: 247572-271811 bp; Ensembl Fugu v12.2.1), and Tetraodon (Tetraodon virtual contig 1) genomic sequences containing either the PRNP or stPrP-2 gene, its whole upstream intergenic sequence, and adjacent genes (gene for PrP-like protein in fishes, PRND in mammals, PRNT in human, and RASSF2 and SLC23A1 in both mammals and fishes). Prior to submission to VISTA, transposable elements in the mammalian sequences were masked by using the RepeatMasker as above. Human sequence and its annotation was used as the base sequence. Pairwise sequence comparisons were calculated with a threshold of 50% identity in a 50 bp window. The minimum identity shown in the VISTA plots is 30%.

3.1.4.1.2 Alignment of SPRN Genomic Context between Mammals and Fish

I aligned human (chr10: 134322741-134374165 bp; Ensembl human v12.31.1), mouse (chr7: 130335510-130371625 bp; Ensembl mouse v12.3.1), rat (chr1: 199669758- 199704156 bp; Ensembl rat v11.2.1), zebrafish (assembly_203: 4494411-4519077 bp; Ensembl zebrafish v14.2.1), Fugu (chr_scaffold_28: 384496-394338 bp; Ensembl Fugu v12.2.1), and Tetraodon (Tetraodon virtual contig 2) genomic sequences containing the SPRN gene, its whole upstream intergenic region, and adjacent genes (gene encoding GTP-binding protein in all species, amine-oxidase-coding gene in mammals and pufferfish, and long-chain fatty-acyl elongase-coding gene in zebrafish). Prior to submission to VISTA, transposable elements in the mammalian sequences were masked by using the RepeatMasker as above. Human sequence and its annotation was used as the base sequence. Pairwise sequence comparisons were calculated with a threshold of 50% identity in a 50 bp window. The minimum identity shown in the VISTA plots is 30%.

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3.1.4.1.3 Alignment of Tammar Wallaby, Human, Mouse, Bovine and Ovine PRNPs

I aligned in silico genomic sequences harbouring the PRNP gene. The 66.5 kb tammar wallaby BAC sequence contained the PRNP gene (30055 - 50234 bp) together with flanking sequences. The human (chr20: 4558866 - 4650555 bp; Ensembl human v16.33.1) and mouse (chr2: 132052342 - 132080812 bp; Ensembl mouse v22.32b.1) genomic DNA sequences contained complete proximal and distal intergenic regions. PRNP in the bovine genomic sequence (AJ298878; NCBI) encompassed 49430 - 69659 bp. I merged two overlapping ovine genomic sequences (U67922 and AY184242; NCBI) into a single 46955 bp contig with the PRNP lying between 5666 - 26295 bp. This sequence contains the complete distal intergenic region (20961 bp). Transposable elements (but not simple repeats and low complexity DNA) in genomic sequences were masked using the RepeatMasker program as above. I used the human sequence and its annotation from the Ensembl genome browser as the base sequence. Pairwise sequence comparisons were calculated with a threshold of 75% identity in a 100 bp window. The minimum identity shown in the VISTA plot is 40%.

3.1.4.2 Local Alignments of Long Genomic Sequences

Using default settings of the PiPMaker program available as a web service (Schwartz et al. 2000; http://bio.cse.psu.edu/cgi-bin/pipmaker?basic), I aligned the Fugu and Tetraodon genomic sequences containing the stPrP-2 coding gene (Ensembl Fugu v12.2.1 chr_scaffold_155: 247572-271811 bp and Tetraodon virtual contig 1 (Chapter 5.4.4) and SPRN (Ensembl Fugu v12.2.1 chr_scaffold_28: 384496-394338 bp and Tetraodon virtual contig 2 (Chapter 5.4.6); and human (chr20: 4657104-4708670 bp; Ensembl human (v12.31.1) and mouse (chr2: 132957930-132998136 bp; Ensembl mouse v12.3.1) genomic sequences bordered by the PRND and RASSF2 genes. Results are presented as percentage of identity (PiP) plots and dot plots.

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3.1.5 Phylogenetic Footprinting

3.1.5.1 Phylogenetic Footprinting of SPRN

I used the FootPrinter program as a web service (Blanchette and Tompa 2002; http://abstract.cs.washington.edu/~blanchem/FootPrinterWeb/FootPrinterInput.pl) to perform phylogenetic footprinting of the human, mouse, and Fugu genomic sequences containing the SPRN gene. In this experiment I used sequence upstream to the SPRN ORF and the whole intergenic region. Using a conservative approach in this analysis, I accepted only motifs detected in all three species and with low parsimony scores: score 0 for 6 and 7 bp motifs, score 1 for 8 and 9 bp motifs, score 2 for 10, 11, and 12 bp motifs. A motif of 13 bp (score 2) was identified after manual inspection of the 12 bp motif sequences.

3.1.5.2 Phylogenetic Footprinting of PRNP

I performed phylogenetic footprinting of the tammar wallaby, human, mouse, bovine, and ovine genomic sequences harbouring PRNP described above using the free FootPrinter program (Blanchette and Tompa 2002; http://abstract.cs.washington.edu/~blanchem/FootPrinterWeb/FootPrinterInput.pl) run locally under the operating system Red Hat Linux ver. 2.4.18-3 (www.redhat.com). This program can detect small conserved motifs in a range 4-16 bp. I used a conservative approach in this analysis accepting only motifs detected in all five species. I allowed 2 bp mismatch for the 13 bp and 12 bp motifs (parsimony score 2), 1 bp mismatch for 11 bp and 10 bp motifs (parsimony score 1), and no mismatch for 9 bp and 8 bp motifs (parsimony score 0). Before phylogenetic footprinting, transposable elements and exons were masked and excised from the sequences. Analyses of the upstream PRNP promoter and PRNP intron were run separately. Due to the limited size of the proximal intergenic sheep sequence (see above), I limited analysis of the upstream promoter to a final sequence size of approximately 5 kb. Because of the large intron sizes, I restricted the search for motifs in the intron to a length of 10 % of the human PRNP intron size (approximately 1270 bp), using an option subregion_size

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1270, as suggested by Blanchette and Tompa (2002). I used the FootPrinter options triplet_filtering and post_filtering in all analyses.

3.1.6 Prediction of Transcription Factor-Binding Sites

Potential transcription-factor-binding sites in all the genomic sequences were identified using the MatInspector program available as a free web service (Quandt et al. 1995; http://www.genomatix.de/). Motifs predicting transcription-factor-binding sites deposited in the TRANSFAC database (Wingender et al. 1996) were identified using this program. I again used a conservative approach in this analysis: motifs were identified with core similarity 1 and matrix similarity score above the optimised matrix similarity score.

3.2. Experimental Methods

3.2.1 PCR, Cloning and Sequencing of PCR Products

Genomic DNA and BAC DNA were isolated following the standard protocols (Sambrook et al., 1989). Plasmid DNA was isolated using the QIAprep Spin Miniprep kit (Qiagen), and RNA was isolated using the RNeasy Mini Kit (Qiagen).

3.2.1.1 Cloning of Tetraodon SPRN ORF

The low quality of the FS_CONTIG_4144_1 genomic sequence did not allow translation of the SPRN ORF so I used its sequence to design PCR primers TeShoF and TeShoR (Appendix II, Table II.1) flanking the SPRN ORF, using the MacVector 7.0 program (Oxford Molecular Group 2000). I extracted DNA from Tetraodon cells (provided by Dr. Frank Grutzner, RSBS) using standard procedures (Sambrook et al. 1989), and used it as the template for PCR amplifications (200 ng per reaction) using the FastStart Taq polymerase kit (Roche) following the manufacturer’s instructions for the PCR procedure using GC-RICH solution. The PCR product was cloned using the TOPO cloning kit (Invitrogen) following the manufacturer’s instructions. I sequenced

111 Chapter 3 Materials and Methods the cloned fragment by using universal primers M13F and M13R (Appendix II, Table II.1) and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer’s instructions. Products of cycle sequencing reactions were run on an ABI3730 DNA sequencer (Applied Biosystems) in Biomolecular Resource Facility (The John Curtin School of Medical Research) as part of commercial service.

3.2.1.2 Cloning of Tammar Wallaby PRNP cDNA

By applying a strategy of Simonic et al. (1997) I developed a new method to screen cDNA libraries using PCR consisting of two steps. First, using genomic DNA as a template I performed PCRs in a total volume of 50 µl containing 1 X PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2; Roche), 200 µM dNTPs (Roche), 1-2 U of the Taq polymerase (Roche), 200 ng of template, and 200 pmol of degenerate primers G-Forward and G-Reverse, respectively (Appendix II, Table II.1). After 2 min of denaturation at 94 ºC, I ran 35 amplification cycles using a touch-down protocol as follows: 1 min of denaturation at 94 ºC, 1 min of annealing with temperatures ranging from 59 ºC to 52 ºC, and 1 min of extension at 72 ºC. Finally, I extended the PCR products further during 10 min at 72 ºC. The 214 bp PCR product was cloned using the pGEM-T Easy (Promega) cloning kit following the manufacturer’s instructions. The plasmids harbouring the cloned fragment were templates for sequencing reactions using the standard primers T3 and T7 (Appendix II, Table II.1) and BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems) following the manufacturer’s instructions. Products of cycle sequencing reactions were run on an ABI3730 DNA sequencer (Applied Biosystems) in Biomolecular Resource Facility (The John Curtin School of Medical Research) a part of commercial service.

In the second step I screened a random primed pouch young tammar wallaby cDNA library as template using PCR. This library was made previously using the ZAPII vector (Clontech) and a female (day 0) and a male (day 0) tammar wallaby pouch young as mRNA sources. Its titre was 109 pfu / ml. After two extension steps in the reverse direction, and after four extension steps in the forward direction, I extended the initial 215 bp to a final length of 1978 bp. Each extension step consisted of two rounds of

112 Chapter 3 Materials and Methods amplification using semi-nested PCR. In round one, I used 1 µl of cDNA library as template with 50 pmol of upstream specific primer and 50 pmol of universal primer (T3 or T7 (Appendix II, Table II.1). In round two, I used 1 µl of the first round PCR reaction as template with 50 pmol of downstream specific primer and 50 pmol of universal primer. The composition of the PCR reaction mixture was as above. The initial denaturation step was 7 min at 94 ºC when the cDNA library was used as template, or 1 min at 94 ºC when the PCR reaction was used as template, followed by 35 amplification cycles of 1 min denaturation at 94 ºC, 1 min of annealing at 65 ºC, and 2 min of extension at 72 ºC. Finally, I extended PCR products for 10 min at 72 ºC. The PCR products amplified in the second round of each extension step were cloned using either the TOPO cloning kit (Invitrogen) or pGEM-T Easy (Promega) cloning kit following the manufacturer’s instructions, and sequenced as above. After each extension step I merged sequences, extended cDNA sequence, and designed new PCR primers.

3.2.1.3 Cloning of Monodelphis domestica PRNP cDNA 3’ end and Assembly of ORF

I first amplified, cloned and sequenced a specific region of Brazilian opossum PRNP CDS using genomic DNA as a template and the degenerate primers G-Forward and G- Reverse as above. In order to amplify the 3’ end of Brazilian opossum PRNP cDNA I screened a poly-T primed Monodelphis testis cDNA library (gift from Dr. Denise Carvalho-Silva, RSBS) made previously using the predigested Lambda ZAP II/EcoR I/CIAP vector (Stratagene) using PCR. In the first step of asymmetric PCR I used 1 µl of library as a template, 50 pmol of primer MdF1 (Appendix II Table II.1) and 2.5 pmol of primer T7 with the same PCR conditions as above. In the second PCR step, I used 0.1 µl of the first step PCR reaction as template and 50 pmol of MdF1 and T7 primer each following the same reaction conditions as above. Finally, I cloned and sequenced the PCR products as above. The sequence (575 bp) contained part of the PRNP ORF between 1-360 bp (ORF coordinates 451-810 bp) and the mRNA 3’ UTR region between 361-575 bp.

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Using the tammar wallaby PRNP ORF nucleotide sequence as search query and the local BLAST service, I searched the Monodelphis Whole Genome Shotgun trace database (NCBI) and identified traces 334046496 and 346363827 corresponding to the Brazilian opossum PRNP ORF regions between 1-470 bp and 1-333 bp, respectively. I combined this information with our experimental data and assembled a complete ORF of 810 bp. Finaly, I merged both ORF and 3’UTR into a single sequence of 1025 bp.

3.2.1.4 PCR using BAC DNA as Template

I used the tammar wallaby PRNP BAC clone DNA as template (100 ng per PCR reaction) and 50 pmol of each G-Forward and G-Reverse primers (Table II.1) as above to test whether BAC clone contains PRNP gene. I then cloned and sequenced 214 bp PCR products as above.

3.2.2 Expression of Tammar Wallaby PRNP

I extracted RNA from snap-frozen tammar wallaby cerebral cortex, testis, mammary gland, liver, kidney, and stomach tissue samples, respectively, using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. I then used 1 µg of RNA as template and random hexamer primers in order to synthesize the first-strand cDNA by using the Expand Reverse Transcriptase kit (Roche), following the manufacturer’s instructions. Next, I designed PCR primers spanning the tammar wallaby PRNP intron (5’RT-F and 5’RT-R, Appendix II Table II.1), and used 5 µl of the first-strand cDNA as template for the PCRs with 25 pmol of each primer. After 5 min of initial denaturation at 94 ºC, I ran 30 amplification cycles with 45 s of denaturation at 94 ºC, 30 s of annealing at 54.5 ºC, and 30 s of extension at 72 ºC. I further extended PCR products for 10 min at 72 ºC. Composition of the PCR reaction mixture was as above. The 185 bp PCR products were cloned and sequenced as above to confirm their identity.

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3.2.3 Screening of Tammar Wallaby Genomic DNA BAC Library

The tammar wallaby bacterial artificial chromosome (BAC) was constructed previously by cutting the genomic DNA from tammar wallaby male adult white blood cells with restriction enzyme HindIII, and ligating the restriction fragments into a pRazorBAC vector (Victorian Institute of Animal Science, Sankovic et al., unpublished). There are 55296 clones in the library with an average insert size of 108 kb and with calculated redundancy of approximately 2.2 times. My probe was the specific 214 bp product from tammar wallaby PRNP amplified as above. I first labelled 25 ng of the probe with 32P- dATP using the MegaprimeTM DNA labelling kit (Amersham) and removed unincorporated 32P-dATPs using the ProbeQuantTM G-50 Micro Columns (Amersham). Next, I prehybridized Hybond-N nylon membranes (Amersham) containing complete BAC library overnight at 65ºC in a modified Church and Gilbert buffer (0.25 M

NaHPO4, 5mM EDTA, 7% SDS, pH 7) containing 100 µg/ml of denatured salmon sperm DNA and 1% of bovine serum albumin (MP Biomedicals). I then added probe and hybridised it to the membranes at 65ºC overnight. After rinsing filters in 2 X SSC and washing them in 2 X SSC / 0.1% SDS, and in 1 X SSC / 0.1 % SDS, I exposed them to an X-ray film (Kodak) for one week.

3.2.4 Restriction Analysis of BAC DNA

I cut the BAC clone DNA with restriction enzymes SalI (Roche), NotI (Roche), XboI (Roche), and both SalI and XboI, respectively. Restriction digests were electrophoresed on a 1% UltraPureTM Agarose (Invitrogen) gel. I determined sizes of the restriction fragments and estimated size of the whole BAC DNA insert (≈70 kb).

3.2.5 Southern Blotting of BAC DNA

I transferred the restriction digests (see above) to the Hybond-N+ (Amersham) nylon membranes according to the standard protocol (Sambrook et al., 1989). I used the same probe as above, and I also labelled it, hybridised it to membranes, and later on washed

115 Chapter 3 Materials and Methods the membranes as above. Finally, I exposed the membranes to the X-ray film (Kodak) overnight.

3.2.6 Fluorescent in situ Hybridisation

I labelled the BAC clone DNA with biotin-14-dATP by nick translation following the BIONICKTM Labelling System kit (Invitrogen) protocol. I then co-precipitated 10 µg of labelled BAC DNA with 10 µg of sheared tammar wallaby genomic DNA and 10 µg of sheared salmon sperm DNA overnight at -20 ºC, dissolved these precipitates in 50% formamide, 10% dextran sulphate and 2 X SSC, denatured them at 80 ºC, pre-annealed them at 37 ºC, and finally applied them to pre-treated slides containing tammar wallaby chromosome metaphase spreads. After three days in moist chamber at 37 ºC, I washed the slides in buffers containing 50 % formamide / 2 X SSC, 2 X SSC, and 0.1 X SSC, and then I blocked them in 5% BSA (MP Biomedicals) with 0.1% Tween 20 (SIGMA). Next, I applied FITC-conjugated avidin and Cy3-conjugated antidigoxin antibodies dissolved in 1% BSA (MP Biomedicals), 0.1% Tween 20 (SIGMA), and 4 X SSC to the slides during 30 min at 37 ºC. After post-antibody washes I stained the slides with DAPI (1 µg/ml in 2 X SSC) and mounted them using the VECTASHIELD (VECTOR) mounting medium.

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