Collection of Skin Biopsies and Blood

Skin biopsies were collected from uncoloured (white) and coloured (black and brown) animals of the merino sheep using disposable, sterile, biopsy punch (8 mm diameter), treated and stored in RNAlater (Sigma-Aldrich, Milan, Italy), transferred to the molecular biology laboratory and immediately frozen in liquid nitrogen until RNA extraction. Blood samples were collected from the jugular vein of the same individuals with PAXgene Blood DNA Tubes (PreAnalytix kit, Qiagen, Milan, Italy) via standard phlebotomy technique, processed immediately in the lab according to the manufacturer’s protocol for the DNA isolation and the aliquotes were stored at -80^oC. Samples were collected and recorded according to the farm technicians from Aziende la Campana Montefiore dell’Aso (Ascoli Piceno, Marche), La Meridiana Umbertide (Perugia, Umbria), Italy with permission from the owners of each farm.

RNA and DNA Isolation and Quantification

Total RNAs were extracted from the stored skin biopsies of all three animals using TRI Reagent (Sigma-Aldrich, Milan, Italy) according to the manufacturer’s instructions followed by treatment with RNase-free DNase (Fermentas, Milan, Italy) to remove contaminated DNAs. Tissue was homogenized (0.075 g in 750 µl TRI reagent) using Polytron homogenizer (Qiagen, Milan, Italy). Genomic DNAs were isolated from the blood samples with PAXgene Blood DNA kit (PreAnalytix kit, Qiagen, Milan, Italy) following the given handbook protocol.

The qualitative assessment of the isolated, purified DNAs, RNAs were done utilizing the Genesys 10 UV Spectrophotometer (Thermo Electron Corporation, Madison, USA). The purity was assessed by calculating the ratio of optical density (OD) at A260/A280 and the integrity was determined by running the samples on 1.0% formaldehyde-agarose gel electrophoresis for RNA and 0.8% agarose gel electrophoresis for DNA [40]. For DNA, the concentration was also evaluated based on the intensities of band with reference to the molecular weight standard Lambda (λ) DNA EcoRI HindIII digest (Fermentas, Milan, Italy) or 1 kb gene ruler (USB Corporation, Cleveland, USA). The nucleic acid concentration was calculated following [40] and the DNA samples were diluted to 10 ng/µl or 50 ng/µl for PCR amplification.

cDNA Synthesis and RT-PCR Amplification

cDNAs were synthesized from total RNA extracted from the skin of the merino sheep. Reverse Transcription (RT) from 1–1.5 µg of RNA in a toal volume of 20 µl containing 50 pmol oligo(dT) (18-mer) or oligo(dT)₁₈ modified primer, 0.5 mM deoxyribonucleoside triphosphate (dNTPs), 1×RT buffer, 20 U of RNase inhibitor and 200 U PrimScript™ Reverse Transcriptase (Takara Bio Inc., Clontech, Jesi, Italy) or StrataScript™ Reverse Transcriptase (Stratagene, Agilent Technologies, Milan, Italy) according to the manufacturer’s instructions. The reaction was incubated for 60 min at 42°C and then heated at 70°C for 15 min, and cooled on ice. All the RT reactions were performed in a Perkin-Elmer Cetus Model 480 DNA Thermal Cycler (Perkin-Elmer, Monza, Italy) and/or MyCycler™ Thermal Cycler (Bio-Rad Laboratories, Segrate, Italy). Subsequently, 0.5–0.7 µl of the first strand cDNA reaction was used for PCR amplification. The reactions were performed in 25 µl volume containing 1×PCR bufffer, 1.5 mM MgCl₂, 2.0 mM dNTPs, 0.3–0.5 µM gene specific primers (Table S2), 20–30 ng/ul cDNA and 1.5 U of proofreading Easy-A High-Fidelity PCR Cloning Enzyme (Stratagene, Agilent Technologies, Milan, Italy) and the cDNA check amplification was performed with Dream Taq DNA polymerase (Fermentas, Milan, Italy). Three-step RT-PCR amplification was performed in a MyCycler™ Thermal Cycler (Bio-Rad Laboratories, Segrate, Italy), TGRADIENT Thermocycler (Biometra GmbH, Göttingen, Germany) with an initial denaturation at 95°C for 3 min, followed by 5 primary cycles of 94°C for 1 min, annealing temperature (Ta^oC) below 3–5°C of the temperature melting (Tm) of the gene specific primer whichever is lowest of the two primers for 1 min, and a 72°C for 1 min. This was then followed by 25 consecutive cycles of 94°C for 15–30 sec, annealing temperature (Ta^oC) for 15–30 sec and 72°C for 20–30 sec with a final extension at 72°C for 10 min, lastly a hold temperature at 4°C. NOTE: PCR cycling conditions especially Ta, timing interval varies with primer sets and the expected size of amplicons (see Table S2 for details).

The isoform (+) specific primer pair for the the amplification of the open reading frame (ORF) corresponding to the 621 bp of the sheep SCF cDNA was designed based on the coding sequence homology among human (GenBank Acc. No. NM_000899.3), chimpanzee (XM_509255.2), marmoset (XM_002752832.1), orangutan (XM_002823566.1), mouse (NM_013598.2), rat (NM_021843.3), panda (XM_002921694.1), cat (NM_001009343.1), horse (NM_001163962.1), dog (NM_001012735.1), goat (AB002152.1), pig (NM_214269.2), cow (NM_174375.2) and sheep (GU386372) using Primer3 software [41]. The remaining 5′ and 3′ RACE SCF gene specific primer pairs were deduced from the 621 bp cDNA coding sequence (CDS) fragment to walk up and down in order obtain the full-length cDNAs. All the designed primer pairs were checked with the online software tools [42], [43] before making an order with [42]. The primers used in this study were synthesized and purchased from Sigma-Aldrich, Milan, Italy.

Rapid Amplification of cDNA end Experiments (5′ and 3′ RACEs)

We performed 5′ and 3′ RACE experiments to isolate and determine the sheep full-length SCF cDNA(s). This was done following the instructions of 5′ (v. 2.0) and 3′ (v. E) RACE System for Rapid Amplification of cDNA Ends (Invitrogen, Life Technologies, Monza, Italy).

5′ RACE cDNAs were reverse transcribed from 1–1.5 µg of RNA in a total volume of 20 µl containing 2–2.5 pmol SCF gene specific splice variant primers (Table S2), 0.5 mM dNTPs, 1×RT buffer, 20 U of RNase inhibitor, 200 U of PrimScript™ Reverse Transcriptase (Takara Bio Inc., Clontech, Jesi, Italy) and StrataScript™ Reverse Transcriptase (Stratagene, Agilent Technologies, Milan, Italy) according to the manufacturer’s instructions. The reaction was incubated for 60 min at 50°C and then heated at 70°C for 15 min, cooled on ice and stored at −20°C. Two different 5′ RACE cDNAs were synthesied with gene specific primer for the SCF (+) and (−) form (see Table S2 for details). This was then followed by 0.1 volume of 3 M sodium acetate, pH 4.8 or 5.2 salt and 2.5 volume of 100% ethanol preicipitation to the final volume of 100 µl 5′ RACE cDNA. The precipitation was carried out at −80°C over night and centifuged twice at 16,000 g for 30 min. The pellet was washed twice with 70% ethanol at 16,000 g for 15 min. The collected, air dried pellet was finally dissolved in 40 µl DEPC treated water and stored as aliquotes at −80°C. A homopolymeric tail was then added to the 3′-end of the purified cDNA (10 µl) using 30 U terminal deoxynucleotidyl transferase (TdT, USB Corporation, Cleveland, USA and Invitrogen, Life Technologies, Monza, Italy) and 0.2 mM dCTP (Fermentas, Milan, Italy) following the protocol of 5′ RACE System (v. 2.0, Invitrogen, Life Technologies, Monza, Italy). The reaction was incubated at 95°C for 3 min for the denaturation and 37°C for 12 min for the addition and then heat inactivated at 70°C for 10 min, cooled on ice and stored at −20°C. Subsequently, 3 µl of the dC-tailed cDNA was used in a final volume of 50 µl for the first round enrichment PCR amplification followed by second round nested amplification using 2–3 µl of the primary enriched RT-PCR reaction. The primer combinations used were adapter forward primers aapfwd (first round), auapfwdnst (second round, nested) and scfrev1 (proteolytic site, + form, first round), scfrev2 (common region, - form, first round) and scfrev3 (common, second round) as the reverse primers, respectively. NOTE: Forward adapter primer sequences were retrieved from the 5′ RACE kit, Invitrogen, Life Technologies, Monza, Italy and synthesised by Sigma-Aldrich, Milan, Italy. The PCR amplification was carried out as described above for 36 cycles and the cycling conditions especially Ta, timing interval varies with 5′ RACE primer sets and the expected size of amplicons (see Table S2 for details).

First strand 3′ RACE cDNAs were prepared with a high Tm oligo(dT)₁₈ modified primer as described above and 1 µl of this cDNA was used in a final volume of 50 µl for the first round PCR amplification. Successive nested, splice variant specific amplifications were performed in a 50 µl PCR volume using 1 µl of the primary enriched RT-PCR reaction.

The PCR was run for 36 cycles as described above and the cycling conditions especially Ta, timing interval varies with specific 5′ and 3′ RACE primer sets (see Table S2 for details). For 3′ RACE the primer pairs having high Tm were subjected to a two-step PCR with a coupled annealing, extension at 69 or 72°C for 3 min 10 sec up to 10 min. The primer combinations used for distinctive 5′ and 3′ RACE amplification and the expected size of amplicons were presented in Table S2. NOTE: Forward adapter primer sequences were retrieved from the 5′ RACE kit, Invitrogen, Life Technologies, Monza, Italy and synthesised by Sigma-Aldrich, Milan, Italy.

DNA Splice Junction Amplification

Blood genomic DNA was amplified to confirm the splice site premature termination with a poly A signal detected on sheep SCF cDNA transcripts. The Expand Long Range, dNTPack (Roche S.p.A., Milan, Italy) was used following the manufacturer’s instructions, including 0.3–0.5 µM specific primers scffwd3 (exon 5, common) and scfrev1 (exon 6, + form specific) (Table S2), 500 µM dNTP mix, 3% DMSO, 100–150 ng of genomic DNA and 3.5 U of Expand Long Range Enzyme mix in a final 50 µl PCR volume. The PCR protocol was performed as per Roche’s kit protocol. Since the available unfinished draft reference sheep genome, Oarv2.0 (current version, March 2011 - till date, http://www.livestockgenomics.csiro.au/sheep/oar2.0.php) did not provide much information regarding the SCF gene, the reference SCF genomic locus at the exon 5-intron (5)-exon 6 splice junction was covered in comparison to the orthologous SCF gene assembly of human, mouse, cow and dog.

Expression of Ovine SCF in Skin

To determine the relative abundance of the SCF (+) and (−) cDNA transcripts, we performed semi-quantitative RT-PCR amplification using two different sets of splice variant specific (+ and -) primers as summarised in Table S2. Four sets of primer pair included (+) form specific forward (exon 5-exon 6: stpro3’Rfwd1), reverse primer (exon 6: scfrev1); the common forward primers (scffwd1, scffwd4) located on the common region of the CDS and a (−) form specific reverse primer (scf(−)rev) which was designed spanning into the exon 7-exon 5 splice junction. Total RNA of 1.5 µg of each animal (white, black, brown) was reverse transcribed into cDNA using 200 U PrimScriptTM Reverse Transcriptase (Takara Bio Inc., Clontech, Jesi, Italy) and 50 pmol oligo(dT) modified primer in a 20 µl reaction volume, as described above. PCR amplification was performed using 0.5 µl of the each cDNA sample as a template in 25 µl of a reaction mixture consisting of 1×DreamTaq buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.5 µM each of primer and 1.5 U of DreamTaq DNA polymerase (Fermentas, Milan, Italy). After an initial denaturation step of 3 min at 95°C, a 3-step PCR programme was carried out with 5 successive cycles of 25 sec at 95°C for denaturation, 25 sec at primer-specific annealing temperature (Ta°C) for annealing procedure and 25 sec at 72°C for extension, followed by 25 repeat cycles of 94°C for 15 sec, annealing temperature (Ta^oC, see Table S2) for 15 sec and 72°C for 20 sec with a final extension at 72°C for 10 min and a cooling phase at 4°C. Amplified RT-PCR products were separated on 1.5–2% agarose gel electrophoresis, and were evaluated by ethidium bromide staining and UV transillumination. For the RT-PCR reference, constitutively expressed glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 252 bp) and 18 S rRNA (132 bp) was used as an equal loading control. The house keeping gene (HKGs) primers were designed from the corresponding Ovis aries NCBI GenBank Accession Nos. (see Table S2) and amplified with the same PCR conditions and cycle numbers. Amplicons were confirmed by cloning and direct sequencing. The relative signal strength was measured using the QuantiScan Demo software [44].

For Northern blot analysis, total RNA was isolated from skin as described above. The poly(A)+ mRNA from total RNA was purified using Oligotex mRNA Midi Kit (Qiagen, Milan, Italy) following the manufacturer’s protocol. The 40 µl eluted mRNA sample was separated on a 1.2% denaturing formaldehyde-agarose gel electrophoresis [40]. Subsequently, mRNA was transferred to a Hybond™-N Neutral nylon membrane (Amersham Biosciences, GE Healthcare Europe GmbH, Milan, Italy) overnight by capillary diffusion [40]. The mRNA was crosslinked onto the membrane by baking at 80°C for 2 h. The membrane was pre-hybridized at 50°C for 1 h and then hybridized overnight at 50°C containing denatured DIG-labeled PCR probe (2 µl/ml). DIG-labeled PCR probes were synthesized using a PCR DIG Probe Synthesis kit (Roche S.p.A., Milan, Italy). DIG labeled DNA fragments of ovine SCF (222 bp, +/− form) and 18 S rRNA (132 bp) were synthesized by PCR using the corresponding cDNA clones as templates and gene-specific primers (see Table S2). Following low (2×5 min with 2×SSC, 0.1% SDS at room temperature) and high (2×15 min with 0.1×SSC, 0.1% SDS at 50°C) stringent washes, the nylon membrane was incubated in the blocking solution for 45 min followed by additional incubation with a blocking solution that contained a 1∶5,000 dilution of alkaline phosphatase conjugated, anti-DIG antibody (Roche S.p.A., Milan, Italy) and incubated for 15–45 min at room temperature. The hybridized probe was detected with the chemiluminescent substrate, CSPD (Roche S.p.A., Milan, Italy). Hybridization signals were detected by exposure of the membrane to Kodak® BioMax® XAR Film (Sigma, Milan, Italy) at room temperature. Pre-hybridization, hybridization, blocking and washing solution recipes were prepared and followed according to the procedures for nonradioactive (DIG) labeling and detection of nucleic acids (Roche S.p.A., Milan, Italy). Probes were stripped at 80°C for 2×60 min before rehybridization according to the manufacture’s instructions (DIG application manual, Roche S.p.A., Milan, Italy).

Gel Electrophoresis and Photography

Amplified products were subjected to 1.2–1.5% agarose gel electrophoresis using 1×TAE buffer (40 mM Tris-acetate, pH 8.0, 1 mM EDTA) at 5–7 V/cm. The gels were stained with 0.5 mg/ml ethidium bromide, visualized on ultraviolet transilluminator (Macrovue model 2011, LKB Produkter, Bromma, Sweden). Gels were captured and analyzed using Kodak Digital Science DC40, 1D software for Electrophoresis Documentation and Analysis System (Kodak, Rochester, New York, USA).

Cloning and Sequencing

All the selected amplicons were gel purified either manually by salt precipitation or using Nucleospin columns (Macherey-Nagel, GmbH & Co. KG, Düren, Germany). Cloning was performed in the TA cloning system (pGEM®-T Easy, Promega, Milan, Italy; pCR®2.1 TOPO, Invitrogen, Life Technologies, Monza, Italy; InsTAclone™, Fermentas, Milan, Italy and pSC-A, StrataClone-UA, Stratagene, Agilent Technologies, Milan, Italy). The ligated products (3–5 µl) were transfered by heat shock treatment into a chemically competent DH5α cells which were prepared manually [40], except for pSC-AStrataClone-UA vector system for which StrataClone SoloPack competent cells were used (included in the kit package). Clones were screened by M13 colony PCR amplification. Identified positive colonies were inoculated into the selective antibiotic LB or SOB medium for the over night culture at 37°C, 150 rpm in a shaker waterbath. Subsequently, plasmid DNAs were isolated [40] and screened for the release of expected insert(s) by analytical single or double restriction enzyme digestion (EcoRI or EcoRI+HindIII) according to the vector map. Positive clones were prepared for sequencing and sequenced by the commercial vendors (StarSEQ, Mainz, Germany; BMR sequencing, Padova, Italy) with M13 forward and/or reverse primer or sequenced with any one of the gene specific primer for deeper sequencing of the inserts whenever necessary. Sequences were viewed with sequencing chromatogram trace viewer FinchTV v. 1.4.0 [45].

Sequence Data

Our new sequenced data of SCF can be accesed through NCBI GenBank accession nos. GU386371– GU386374 (Table S1).

mRNA Secondary Structure Analysis

We used the webserver program Mfold v. 3.5 [46] for predicting the non-coding RNA (ncRNA) secondary structure stability of the different SCF transcripts and its miRNA target binding sites. The structure of DNA splice junction was analysed with DNA Folding Form [46]. The ncRNA secondary structures were also predicted with a set of MUSCLE [47] aligned mammalian homologous sequences of the SCF cDNA transcripts using Sequences Selection for the Comparative Approach (SSCA) by Tfold [48]. The optimal secondary structures for all sequences were obtained in a dot-bracket notation with minimum free energy and the structural elements such as helices, internal and terminal loops were deterrmined by drawing the RNA structure in the java applet VARNA v. 3.7 [49]. All fold analyses were performed using the default setting of the web servers.

The TargetScan program Release 5.1 [50] and miRBase Release 16 [51] were used to locate potential sheep SCF 3′ UTR miRNA target sites from human, mouse, dog, cow and chicken.

Protein Homology Modelling

Protein templates were identified and scrutinized using Template Identification tool at SWISSMODEL Workspace v 8.0.5 [52], Reverse PSI-BLAST (in BLAST 2.2.12 packages) search against protein data bank (PDB) and Structural Classification of Proteins (SCOP) at Genomes TO Protein structures and functions (GTOP) [53].

The homology modeling was performed with Modeller 9v2 [54] using an integrated multiple sequence alignment and multiple structure visualization application ‘Friend’ v. 2.0 [55]. All the modelled structures were stored as a PDB format data (.pdb) and then viewed, edited with ViewerLite v. 5.0, Discovery Studio Visualizer 2.5.5 [56]. Modelled structures were assessed with Protein Structure and Model Assessment Tools at SWISSMODEL Workspace. The secondary structures such as Alpha helix, Beta strand, Beta bulge, 3,4,5-turns were defined with respective colours using CCP4MG release 2.4.3 [57]. Homology modeling was also attempted with an automated modeling server at SWISSMODEL Workspace [52].

Sequence Analysis and Molecular Phylogeny

Whole mammalian genome scanning was done to identify the homologous regions of sheep SCF cDNA transcript variants using Basic Local Alignment Search Tool (BLAST) at National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA [58], ENSEMBL release 60 [59] and BLAT [60] searches, sequentially. Sequences were edited, translated using the BioEdit v.7.0.5.2 (Ibis Therapeutics, Carlsbad, CA, USA) [61] and DNASTAR 7 [62] software packages. The open reading frame (ORF) of the full-length SCF cDNAs was determined by DNASTAR 7 [62] and ORF Finder at NCBI (www.ncbi.nlm.nih.gov/gorf/). The positions of exons and introns were determined and the translated SCF protein to genome structure was drawn using WebScipio [63] in reference to the SCF gene structure of human, mouse and dog. ClustalW2 [64] and MUSCLE [47] programs were used to align the DNA and protein sequences. Subsequently, Gblocks program [65] was used to eliminate the poorly aligned positions and divergent regions on the DNA and protein alignments for the phylogenetic analysis. The datas were then converted to FASTA (.fas) and NEXUS (.nex) formats using DataConvert (v. 1.0) [66].

Distance based neighbour-joining (NJ) phylogenetic trees were generated using the Molecular Evolutionary Genetics Analysis (MEGA) software v. 4.1 [67]. The NJ algorithm [68] was implemented with the p-distance [69], Jukes-Cantor [70] and Tamura-Nei [71], [72] model using a transition+transversion substitution at uniform rates as well with the gamma parameter of 4.0. The robustness of each phylogeny was assessed by percentage of 1000 bootstrap (BS) [73] re-samplings.

Phylogenetic relationships were inferred using Maximum Likelihood (ML) method with the programs PhyML-aLRT (v. 2.4.5) [74], RAxML (v. 2.2.3) [75] using the java application program TOPALi (v. 2.5) [76] and MrBayes (v 3.1.2) [77] for the Bayesian Inference (BI) analyses. Among the 88 models tested, two best models Hasegawa Kishino Yano (HKY) [78] plus gamma (+G) distributed rate heterogeneity, General Time-Reversible (GTR+G) [79] matrices for nucleotides and Jones Taylor Thornton (JTT+G) [80] matrix for protein alignments were chosen and subjected to ML analyses as described above. The topology of the trees was inferred by running 1000 bootstrap replicates and expressed as a percentage.

Bayesian Inference consisted of two independent Markov Chain Monte Carlo (MCMC, mcmc nruns) runs of 100,000 (ngen) were calculated with trees samples at every 10^th generation and with a prior burn-in of 25% (sump burnin = 2500; sumt burnin = 2500) i.e., the first 2500 sampled trees were discarded. BI was run with GTR+G, HKY+G and a JTT+G substitution models under the above set parameters for the nucleotide and amino acids alignments, respectively.

Molecular phylogeny models were selected based on the Akaike Information Criterion (AIC), Akaike Information Criterion corrected verion (AICc), Bayesian Information Criterion (BIC) and/or log Likelihood (-lnL) scores, implemented in jModeltest (v. 0.1.1) [81] for nucleotides and ProtTest (v. 2.4) [82] for proteins. Models selection were also performed and compared with TOPALi (v. 2.5) [76]. All the tree files (NJ, ML, BI) were stored in Nexus (.nex) or New Hampshire Tree (.tre) format. Trees were inspected and prepared in FigTree v 1.3.1 software [83].

Use of other Computational Tools and Databases

Ovine SCF transcripts were searched on chr. 5 of the Bos taurus (Btau_5.2, current release 2011) chromosomal map using NCBI map Viewer [84]. The sequence similarity was visualized with Circos table viewer [85]. The post-transcriptional associated regulatory elements located in the 5′ and 3′ untranslated regions (UTRs) of the SCF cDNA transcripts were retrieved from UTR databases (UTRdb or UTRSite) [86] using the online tools UTRScan and UTRBlast. The graphical representation of SCF amino acid and nucleic acid multiple sequence alignment was drawn by a sequence logo generator, WebLogo (v. 2.8.2) [87]. SCF polyadenylation sites were predicted using the polyADQ web server [88]. Alternative splicing pattern of the ovine SCF transcripts with human, mouse reference assembly were predicted using ACEVIEW [89] and Alternative Splicing and Transcript Diversity (ASTD 1.1) [90]. The splice site prediction such as putative alternative exon isoform, cryptic and constitutive splice sites of internal (coding) exons was performed using Alternative Splice Site Predictor (ASSP) [91] and Regulatory RNA Motifs and Elements Finder (RegRNA Release 1.0) [92]. SCF protein knowledge, sequence analysis, classification were performed with the UniProtKB Protein existence Server [93]. SCF protein secondary structure and site interactions were analyzed using Protein data Bank (PDB) [94] and PDBsum [95]. The putative SCF protein domain figure was drawn with MyDomains - Image Creator at ExPASy [96].

Ethics Statement

In agreement with the new European Directive on the protection of animals used for scientific purposes (Directive 2010/63/EU, Article 15, Annex VIII), all animal procedures used in the study are classified as ‘mild’ (i.e. procedures with no significant impairment of the well-being or general condition of the animals) and have been preemptively approved by the Animal Ethics Committee of the University of Camerino.

Identification and Isolation of the Sheep SCF cDNA Fragment

To examine the SCF variant(s) expressed in the skin of white merino sheep, 1–1.5 µg of total RNAs from the skin were reverse transcribed and the synthesized single strand cDNAs were amplified by PCR. We initially carried out the cDNA coding (CDS) region amplification using the primer pair scffwd1 and scfrev1 (Table S2). Primer walking and the mRNA/cDNA structural coverage of the longer and shorter cDNA amplification strategies from the ovine total RNA (skin) are shown in Figure 1A and 1B. RT-PCR primers were selected based on the mammalian nucleotide (nt) sequence alignment of the soluble-SCF (s-SCF) cDNA encompassed to the open reading frame (ORF) of 606 bp of the 621 bp amplicon (Figure 1A(a)) commonly known as ‘soluble or secreted form’. The purified RT-PCR amplification product was then cloned and sequenced. Sequencing results revealed no differences among white, black and brown clones of the 621 bp (Figure S1A), which additionally appear to be identical (99%) with two of the previously submitted NCBI GenBank mRNA (partial) sequences of ovine s-SCF (U89874.1 in 2002 and Z50743.1 in 2005; see Figure S1B) from ovarian follicles and keratinocytes, respectively. An exception of transition at T⁵⁴C in U89874.1 was observed among the 621 bp sequences. Similarly, a transversion at C⁸¹G was observed (see the chromatogram of Figure S1B) in 2 out of 5 clones sequenced in white animal. The possible allelic variant at this position will elucidate its true identity. Nevertheless, these substitutions do not result in an amino acid substitution change.

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Figure 1. Schematic illustration of primer walking strategy for the ovine SCF (oSCF) mRNA/cDNA transcripts from skin. Gel pictures show subsequent RT-PCR and RACE amplification of the resultant full-length structural coverage of s-SCF (+) and m-SCF (−).

The (+) and (−) product is indicated with two different symbols (see key to symbols). In the above figure, the arrows indicate the corresponding position of fwd and rev primers and the split regions of 5′ UTRs, CDS and 3′ UTRs are labeled with respective positions and base pairs (bp). The start and stop codon is labeled in ‘black’ (ATG) and ‘red’ (TAA) letters repectively. (A) Illustration for the full-length cDNA coverage of ovine s-SCF (+) isofom-1. (a) Amplification of isoform specific coding region (CDS) of ovine s-SCF (+) cDNA fragment (621 bp). Individual animal of merino sheep such as Black, Brown, White and the PCR negative control is indicated as Bl, Br, Wh_1,2 (two individuals) and (−)ve, respectively; (b) 3′ RACE first round amplification of oSCF common region (+/−) showing three different sizes of amplicon ranging from ∼700 bp to 1300 bp; (b₁) Isoform specific second round (Nested 1) 3′ RACE of ovine s-SCF (+) cDNA fragment (855 bp); (b₂) Gel picture shows the purified 3′ RACE product of 793 bp (Nested 2 amplification); (c) 5′ RACE proteolytic site specific amplification (364 bp) of ovine s-SCF (+). (B) Illustration for the full-length cDNA coverage of ovine m-SCF (−) isoform-2. A premature termination codon (PTC) is indicated in red symbol and the resultant alternative open reading frame (ORF) responsible for the shorter truncated product is highlighted in black open box symbol; (b₃) 3′ RACE amplification (Nested-1) from (b) indicates a 597 bp ovine m-SCF (−) amplicon and the other non-specific products (∼0.7/1.2 kb); (b₄) Further, Nested 2 amplification yielded a 389 bp ovine m-SCF (−) amplicon; (b₅) Gel picture shows the Nested 3 amplification of a 336 bp ovine m-SCF (−) product amplified from either (b₃) or (b₄) or (b) or directly from the oligo(dT)₁₈ modified primed cDNA; (d) 5′ RACE amplification of the common region (+/−) showing two oSCF cDNA products (325 and 215 bp) characterized as ovine m-SCF (−) isoform2a/ab, respectively. DNA size markers are indicated as, M₁– λ-DNA EcoRI/HindIII digest; and M₂ - 1 kb Gene Ruler. In the above figure, the arrow marks indicate the appropriate size(s) of amplicon of the respective (RT)-PCR amplification. Note: For simplification we removed the tag ‘scf’ from the primer notation (see. Table S2).

https://doi.org/10.1371/journal.pone.0038657.g001

The virtual translation of the 606 bp CDS (of the 621 bp) resulted in a protein corresponding to the first 202 amino acids (aa) (Figure S1A and S1B) of the ovine SCF (oSCF), in which the last 28 aa at the C-terminus was spanned into the putative primary proteolytic region of the long isoform i.e., s-SCF or (+) form and is identical to three of the GenBank oSCF protein sequences of 260 aa, 202 aa and 267 aa (Acc. No. AAB49491, CAA90620.1 and P79368), respectively.

Rapid Amplification of cDNA Ends (RACE)

To obtain the full length cDNAs, we performed the 3′ and 5′ RACE experiments sequentially. Two different sets of primer (Table S2) were used for RT-PCR amplification in order to ascertain the corresponding 3′ and 5′ untranslated regions (UTRs) of the two different transcript variants i.e., (+) and (−).

3′ RACE – Detection of a Splice Variant of Ovine SCF

Initially, 3′ RACE cDNAs were prepared as described in materials and methods. One µl of this cDNA was used for the first round PCR amplification with the common CDS region forward primer and oligo(dT)₁₈ modified as a reverse primer (Table S2). We got an unexpected short size of approximately 350 bp prominent amplicon since the expected 3′ UTR sequence with respect to other mammalian SCF mRNA species ranges from ∼500 bp to ∼4.5 kb. This was then gel purified, cloned into the TA cloning system and sequenced. To our surprise, the BLASTN sequence analyses revealed a 336 bp oSCF product (Figure 1B(b₅)). Overlapping the 336 bp to the 621 bp CDS amplicon, we obtained a novel, truncated oSCF mRNA splice variant of 691 bp (without the 5′ UTR). Subsequent virtual translation of the ORF containing 546 bp resulted in a truncated oSCF protein of 181 amino acids with a unique C-terminus. The concomitant deletion in the shorter clone resulted in the substitution of aspartic acid (D) at aa pos. 175 with glutamic acid (G) i.e., D¹⁷⁵G. Truncation would delete the C-terminal 93 aa residues of ovine s-SCF and fully conserved till G¹⁷⁵ which is explained below. Henceforth, the new truncated protein isoform has a short stretch of 6 aa sequences right after the ‘G¹⁷⁵’ residue ‘KTYKHS’ as its C-terminus (Figure S1B). This shorter form of oSCF has not been previously reported; however, short isoforms of SCF commonly known as membrane-bound form (m-SCF) corresponding to 245 aa lacking the proteolytic site have been reported as the (−) form of previously reported mammalian species including human [97], [100], mouse [11], cow [98] and avain [99]. The newly identified 181 aa oSCF (−) form differed from the 245 aa by the deletion of 64 aa at the C-terminus corresponding to the transmembrane and intracellular region. Hence, this novel cDNA variant could be recognized as the ‘membrane-anchored’ SCF protein (m-SCF) form and named as ‘SCF truncated isoform-2’, designated hereafter as (−) form since it lacks the primary proteolytic site. To our knowledge, this information of oSCF truncated (−) protein product is previously unreported in other mammal species especially in skin.

The remaining short 145 bp (after removing the adapter sequences from the oligo(dT)₁₈ modified primer) including the polyA nucleotides belong to the 3′ UTR of ovine m-SCF (−) form. Mammalian genome scanning for the SCF gene represented that this novel 3′ UTR of ovine m-SCF (−) form corresponds to the intervening sequence in between exon 5 and exon 6 i.e., intron-5 of the (+) form. Here we hypothesis that the premature truncation could be the result of alternative use of the splice donor/acceptor site in the intervening sequences. Later, this short 3′ UTR amplification was confirmed (Figure 1B(b₅)) in black and brown animals by direct sequencing but did not considered for further characterization such as SNPs.

In order to identify the 3′ UTR of the (+) form, we used the same 3′ RACE cDNA preparation as mentioned above. One µl was used for the first round amplification with the common CDS region forward primer and oligo(dT)₁₈ modified as the reverse primer (Table S2). We obtained three different RT-PCR amplicons ranging from ∼700 to 1300 bp (Figure 1A(b)). At this stage it was difficult to substantiate this amplification. Hence, we performed three individual nested amplification sequentially using oligo(dT)₁₈ modified as the reverse primer with the Nested forward primers (Table S2) for the consequent PCR reactions. All these amplified nested fragments were gel purified, cloned into the TA cloning system. Colonies were screened by colony PCR as well by restriction digestion, and the positive clones were subjected to sequencing.

Sequencing results showed three different sizes of fragment, one each from the Nested amplification (Table S2) viz. 597 bp (Figure 1B(b₃)); 389 bp (Figure 1B(b₄)); and 336 bp (Figure 1B(b₅)) as positives for oSCF. Sequence analysis by BLASTN, BLASTP and ClustalW2 revealed all three products as ovine m-SCF (−) form and are identical to the one described above i.e., 336 bp for the reason that the (−) form override (+) form during the RT-PCR amplification. In other words, there exists a considerable difference in the mRNA expression level between these two transcript variants which is further explained in the later section. The rest of the amplicons were found to be non-specific including the two expected amplicons viz. ∼0.7/1.2 kb (Figure 1B(b₃)) amplified from the primary RT-PCR amplification (Figure 1A(b) and B(b)).

In all the above cases, we obtained always the (−) form, hence we designed a splice variant specific Nested forward primer (Table S2) with higher Tm for the (+) form. The primer was designed in between two exonic junctions (see Figure 1A and 2(c)) spanning into the proteolytic site viz. exon 5 into exon 6 in reference to the human, mouse, dog, horse SCF (source: Ensembl). The second round 3′ RACE amplification (Nested 1; see Table S2) was performed with 1 µl of the primary reaction product using (+) form specific forward primer (Table S2) and oligo(dT)₁₈ modified reverse primer into a final PCR volume of 50 µl. The RT-PCR yielded an amplicon size of 855 bp (Figure 1A(b₁)). Further third round amplification (Nested 2; see Table S2) yielded the expected 793 bp amplicon with some non-specific amplicons. The purified fragment of 793 bp (Figure 1A(b₂)) was then cloned and sequenced. Sequence analyses by BLASTN and BLASTP confirmed the oSCF and named as ‘SCF isoform-1’, hereafter referred as (+) form, which is the counterpart of previously reported ‘soluble’ SCF (s-SCF) sequences in other vertebrate species [99]–[103] (source: GenBank, NCBI). Overlapping and editing of the 793 bp 3′ UTR fragment with the 621 bp CDS fragment, we obtained a total length of 1330 bp (without the 5′ UTR). The ORF of 825 bp corresponding to the deduced amino acid sequence of 274 aa revealed it as the s-SCF (+) form, indicating that this cDNA encodes the ‘soluble’ form of oSCF. This ovine s-SCF (+) form included the stretch of 28 aa recognized as a putative primary proteolytic site (Figure S1B) right after the D¹⁷⁵ at its C-terminus as observed in the previously reported sequences [99]–[103]. The remaining long 505 bp (after removing the adapter sequences from the oligo(dT)₁₈ modified primer) including the polyA nucleotides belong to the 3′ UTR of ovine s-SCF (+) form. The other two amplicons (data not shown) were found to be non-specific and omitted from further characterization.

5′ RACE

To determine the 5′ UTR of the oSCF (+) form, a gene specific 5′ RACE cDNA was synthesized using the proteolytic site specific reverse primer (Figure 1A; see Table S2) as described in materials and methods. Three µl of the dC-tailed cDNA was subjected to the first round RT-PCR amplification with the respective forward and reverse primer (Table S2). After the primary RT-PCR, the expected size of ∼780 bp amplicon was not detected on the gel. Consequently, a second round nested amplification was performed with a common CDS reverse primer and the forward adapter primer (Table S2) using 2–3 µl of the primary enriched RT-PCR reaction. Upon electrophoresis (1.5%), the secondary reaction yielded a single clear amplicon of ∼380 bp as expected. BLASTN results confirmed the sequenced clone of 364 bp (Figure 1A(c)) with the other mammalian s-SCF (+) form and was characterized as ovine s-SCF isoform-1 i.e., (+) form. Overlapping and sequence comparison of this 5′ UTR clone to the 1330 bp (CDS +3′ RACE), resulted in a deduced 189 bp 5′ UTR sequence of ovine s-SCF (+).

The 5′ RACE RT-PCR for the oSCF (−) form was performed in a final PCR reaction volume of 50 µl containing 3 µl of the dC-tailed cDNA which was synthesised by a common CDS reverse primer (Figure 1B; see Table S2) along with all other necessary components as described in materials and methods. The first round enrichment PCR amplification was carried out using the same CDS reverse primer and the forward adapter primer (Table S2). The second round nested amplification was performed with another common CDS reverse primer and the forward adapter primer (Table S2) using 2–3 µl of the primary enriched RT-PCR reaction. Upon 1.5% gel electrophoresis, the secondary reaction yielded two distinct amplicons in the range of 200 to 330 bp. The two amplified 5′ RACE products were gel purified, cloned and sequenced. Sequence analysis revealed the two oSCF 5′ RACE products of sizes 325 bp and 215 bp (Figure 1B(d)). These two 5′ UTR products were not detected in the (+) form specific 5′ RACE cDNA (Figure 1A(c)) though the primer combination rely on the common CDS region. Hence, these two 5′ UTR products were characterized and named as ‘SCF isoform-2a (−) and 2b (−)’, respectively (Figure S4(a₁)). In order to confirm the amplification, this common 5′ RACE was repeated twice along with the (+) form specific 5′ RACE RT-PCR. Overlapping and sequence comparison of these two clones with the 691 bp (CDS +3′ RACE) revealed a deduced 144 bp, a 34 bp 5′ UTR sequences (after subtraction of the forward adapter primer sequence) for the two respective clones (325 bp, 215 bp).

Genomic DNA – Spliceosomal Intron-5 Specific Amplification of oSCF

To verify the alternative splicing (AS) event that resulted in the shorter mRNA transcript i.e., ovine m-SCF (−) form, we amplified the intervening sequence between two exons. The sequenced chromatogram from the cDNA and gDNA of oSCF illustrating a PTC followed by the p(A)_11/18 tail signal is shown in Figure 2(a,b), respectively. The reference SCF genomic locus at the exon 5-intron(5)-exon 6 splice junction was determined in comparison to the orthologous SCF gene assembly of human, mouse, rat, cow, horse and dog (source: Ensembl). The genomic DNA (gDNA) was obtained from the blood of white merino sheep. A expected amplicon size of 948 bp amplicon (Figure 2(d)) was amplified using an exon-5 (common CDS) specific forward primer and exon 6 specific reverse primer (+ form, proteolytic site; Table S2) as shown in Figure 2(c). Sequence analyses and orthologous comparison of the oSCF gene product (948 bp) with other mammals revealed that the first 136 bp corresponds to exon 5, followed by an intron-5 of 729 bp (Figure S4(b)) and an exon 6 containing 83 bp which encodes for the primary proteolytic site. This result was compared with the shorter cDNA transcript. The first 161 nt including a 11 bp polyA (pA) stretch of the intron-5 exhibited 100% identity to the nt pos. 668–835 of the shorter cDNA (Figure S4(c)). However, careful annotation of the 161 nt unveil a premature stop codon at nt pos. 21–23 of the 729 bp intronic sequnce. Figure 3 shows the oSCF gene structure(s) in reference to mouse, dog and human SCF gene (see also Figure S2 for the humanSCF alternative forms). The overall similarity for this 948 bp DNA splice region in other vertebrates was found to be highest with goat and cow SCF (99 and 94%) where as the lowest was detected with chicken and zebra finch SCF (62%).

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Figure 2. Sequencing chromatogram of cDNA in comparison with the gDNA amplification of oSCF gene.

(a) Complementary DNA (cDNA) chromatogram shows the CDS, GT repeats and p(A)₁₈ tail adapter primer as ‘black dotted oval mark’ and a premature termination codon (PTC) as ‘red dotted oval mark’ on the 3′ RACE product (336 bp, see Figure 1B(b₅)); (b) Genomic DNA (gDNA) chromatogram shows the counter part of the above cDNA illustration (a) on exon 5 to exon 6 intervened by intron-5 sequences of the oSCF gene; (c) Amplification scheme of 948 bp splice junction covering exon(5)-intron-5-exon(6) of the oSCF gene with reference to human and mouse. The two exons 5, 6 are differentiated by ‘open and shaded box’ respectively. Arrows over the boxes indicate the fwd and rev primer. Different symbols on the intron-5 indicate the part of retained intronic sequences (161 bp) by a PTC along with the stretch of p(A)₁₁ signal (see key to symbols below the diagram); (d) Gel picture shows the PCR amplification of 948 bp fragment corresponding to the above schema (c) of oSCF gene from blood gDNA; In the picture, arrow mark indicates the exact size of amplicon; M₁ indicates DNA size marker of λ-DNA EcoRI/HindIII digest; and (−)ve represents PCR negative control.

https://doi.org/10.1371/journal.pone.0038657.g002

Intron-5 has a constitutive 5′ splice donor (GT) at its start and six other alternative isoform/cryptic splice donor (GT) sites (Figure S4(b)). Similarly, it has a constitutive 3′ splice acceptor (AG) site exactly at the end of the intron-5 and five other alternative isoform/cryptic splice acceptor (AG) sites (Figure S4(b); see also Figure 3(d)) as predicted by ASSP, RegRNA [91], [92]. Seven important sequences, the so called the ‘branch site’ (BS; Figure S4(b)) viz. CU(Pu)A(Py) are located 20 to 75 bases upstream of the predicted acceptor site. Of which ‘CUGAC’, ‘CUAAU’ and ‘CUGAU’ are considered at most to be the main branch point sites that could be involved in the AS event. PolyADQ [88] prediction revealed two polyadenylation signal (PAS) of the type ‘AAUAAA’ in the 729 bp gDNA (intron-5; Figure S4(b)), but present after p(A)₁₁ stretch hence was not considered to be part of the polyadenylation. However, here we hypothesis that the two other single base variants of ‘AAUAAA’ [104] such as type ‘UAUAAA’ at nt pos. 24 (right after the stop ‘TAA’), 126 and ‘AAUAUA’ at nt pos. 83, 124 found just before p(A)₁₁ bp stretch could be responsible for the polyadenylation process of the shorter mRNA (−) transcript (Figure S4(b); see also Figure 3(d)). These two strong polyA signals are also present in the cDNAs of the respective oSCF mRNA transcripts (Figure S4(c)). The other two single base variants ‘AAUAGA’ and ‘UAUAAA’ detected at 407 nt and 427 nt (Figure S4(b)) away from (pA)₁₁ stretch, respectively, in the same intron, are not considered further the AS analyses.

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Figure 3. Gene architecture of ovine SCF gene in reference to human, mouse and dog.

(a) Schematic representation of human SCF (huSCF) gene is shown. It consists of 10 exons (open boxes) intervened by 9 introns (linear black lines). Regular splicing and polyadenylation generates the full length huSCF mRNA transcript variant-b, a longer (+) form (5460 bp) encoding for a soluble product (273 aa; see Figure 5a); (b) The 84 bp exon 6 encoding for the 28 aa proteolytic site is skipped by an AS event of the huSCF gene is shown. The resultant full length huSCF mRNA transcript variant-a, known as (−) form (5376 bp) encodes for a membrane-bound product (245 aa; see Figure 5b); (c) Schematic representation of ovine SCF (oSCF) gene is shown. Regular splicing of exons 1–9/10(?) generate the full length oSCF (+) mRNA transcript (1519 bp) that encodes for a soluble product (274 aa; see Figure 5a); (d) Conversely, the possible AS events on intron-5 (Ref. human, mouse and dog) resulted in an alternative ORF with a premature termination (red symbol, PTC; see key to symbols). This resulted in retaining of 161 bp intronic sequence and completely eliminating (skipping of) the involvement of exon 6–9/10(?). The deduced protein sequence of this novel, shorter splice transcript variant (835 bp) resulted in 181 aa (see Figure 5b), a membrane-bound product of oSCF (−). In the above illustration, the open square or rectangle box symbolize exon and inverted triangle box symbolize intron. The open and shaded ‘black sparkle’ symbol on exon 10 (in 3a,b), exon ‘?’ (in 3c) and intron-5 (in 3d) all indicate the posssible position of predicted polyadenylation signal (PAS) sites. The two different sizes of the opened ‘black sparkle’ symbol (in 3a,b) denote the frequency of the common PAS such as ‘AAUAAA’ (8–12 times) and ‘AUUAAA’ (4–6 times) in the longer 3′ UTR of human, goat, mouse and rat. In contrast, the shaded ‘black sparkle’ symbol (in 3c,d) represents the other single basse ‘variants’ of PAS (see text in Results). Exon ‘?’ symbol (in 3c) represents the uncertainity of exon 10 position for oSCF (+). A ‘black hook’ symbolize the capped 5′ end and p(A) represents the polyA stretch on the preRNA, mRNA, respectively. The point of transcription termination (TAA) is symbolized as ‘red’ mark on intron-5 of oSCF (−) followed by the illustration of two possible mechanisms that resulted in a PTC of oSCF (−) (in dotted lines). The ASSP predicted constitutive and/or alternative splice donor (GT) and splice acceptor (AG) site(s) are labeled in blue and red letters respectively; (e) Schematic representation of the soluble oSCF (+) gene structure is shown. The exons/introns and the location of non-coding regions are determined in comparison to the mouse (chr 10) and dog (chr 15) SCF gene. The ‘intron (?)’ labeled in blue on the oSCF (+) in reference to mouse chr 10 indicates that the corresponding intron-7 is incomplete at that point i.e., it doesn’t show appropriate 5′ and/or 3′ splice sites; (f) Figure shows the gene stucture of membrane-bound oSCF (−). The ‘black line’ at the end of exon 5 of oSCF (−) in reference to mouse and dog indicate ‘gap’ i.e., coding sequence not found on the respective contig. The ‘vertical red lines’ over the exons indicate ‘mismatch’ of the oSCF (+) and (−) protein with dog (27 aa and 22 aa) and mouse (53 aa and 39 aa) SCF gene.

https://doi.org/10.1371/journal.pone.0038657.g003

Chromosome Location and Genomic Structure of the oSCF (KITLG)

Upon scanning through the sheep genome Oarv2.0 (March 2011 – till date) covering position from 124,495,129 to 124,515,933 of Ovine (Texel) Version 2.0 (current) Genome Assembly we obtained the mere size of OAR3: 20.8 kbp (data not shown). It represents only 19% of the known SCF gene size when compared to human (108.74 kbp), mouse (104.78 kbp), cow (122.28 kbp) and dog (100.21 kbp) (source: Ensembl). The gene encoding the ovine SCF (NCBI gene ID: 443371) is located within a syntenic group on chromosome 3 [105], corresponding to the Sl or kitlg gene locus. This portion of ovine chr 3 is homologous to cattle chr 5. Hence, a comparative chromosomal mapping (Figure 4) was performed at the NCBI Map Viewer [84] of the sheep SCF to the cow SCF i.e., O.ari chr 5 to B.tau chr 5 and O.ari chr 3 to B.tau chr 5. Genomic DNA and cDNA sequence comparison and prediction [63] revealed that the oSCF gene consists of 9 exons interrupted by 8 introns to the dog (Figure 3(e)), pig, horse SCF gene where as in comparison to human, chimpanzee, marmoset, mouse (Figure 3(e)) and rat including the unfinished alpaca genome (source: Ensembl), oSCF gene has been characterized by 10 exons and 9 introns. Comparative analyses of oSCF (+) protein to the dog and mouse SCF gene assembly exhibited 96/90.1 and 93/80.6 match ratio and % identity, respectively. Similarly, oSCF (−) protein showed the match ratio and % identity of 91/87.2 and 90/77.7 to dog and mouse SCF gene assembly, respectively. Among the 9/10 exons, it is predicted by gene annotation (source: Ensembl) that the exon 5 and exon 6 has its importance in determining the final protein product through AS event(s) and the longer exon 10 (9) corresponds to ∼4.4 kb 3′ UTR in human, chimpanzee, mouse, rat and goat in contrast to the shorter 3′ UTR in sheep (reported in this study), cow, pig, horse, dog, cat and panda (source: Ensembl).

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Figure 4. NCBI Map Viewer of ovine SCF (oSCF) aligned to the bovine SCF gene.

The comparative map depicts unknown map region (in red dots) of the oSCF gene to the counter-part of bovine SCF (KITLG) on chr_5:Btau_5.2 displaying regions of 20,582,700–20,618,400 bp. Arrows indicate mapped GenBank records (Acc. No.) with description, respectively.

https://doi.org/10.1371/journal.pone.0038657.g004

Protein Characterization of the Ovine SCF s-SCF (+) and m-SCF (−)

The molecular mass of the oSCF isofoms presented in this study as predicted by EditSeq, DNASTAR [62] is 31.1 kDa and its theoretical iso-electric point is 5.236 for the s-SCF (+) isoform corresponding to the 274 aa. Similarly, the m-SCF (−) isoform has a molecular weight of 20.6 kDa with a theoretical iso-electric point of 6.002 for the 181 aa residues.

Topological features of both the isoforms (+ and −) of ovine SCF in comparison to the human SCF is given in Figure 5(a,b). In ovine s-SCF (+) form, the first 25 amino acids contain features (Figure 5a) of a signal peptide, followed by an extracellular mature chain (aa pos. 26–215), a putative hydrophobic transmembrane region (aa pos. 216–238), and a 35 amino acid intracellular domain (aa pos. 239–274). The 28 aa proteolytic site resides at aa pos. 175–202 which includes a N-linked glycosylation site at aa pos. 196. The four cysteine residues found within the extracellular domain that may result in disulfide bridges viz. (29-s-s-114), (68-s-s-164) and the three other N-glycosylation sites found in the extracellular domain at aa pos. 90, 97, 145 are conserved with all the mammalian s-SCF (Figure S3(d)). The above described features of ovine m-SCF (−) form has been shown in Figure 5(b), which depicts the shortage/deletion of the primary proteolytic site including an N-glycosylation site, a trasmembrane domain (necessary to make a soluble product) and a cytoplasmic domain. The sketch of oSCF gene transcription and translation is shown in Figure 5(c).

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Figure 5. Schematic representation of the topological characteristics of two different ovine SCF (oSCF) protein products in comparison to human SCF (huSCF).

(a) Illustrates the identical topological features for the soluble oSCF (+) and huSCF (+) which corresponds to the 273 aa vs. 274 aa, respectively. The D^174/175G represents the change of aa residue for the alternative natural variant i.e., right at the proteolytic site (28 aa, ‘green line’). The difference in the position is due to sequence divergence of soluble oSCF (+) which has an additional ‘Glu’ residue at ‘E¹⁵⁴’ (see Figure S3d). (b) Demonstrates the difference in topological features of the membrane-bound oSCF (−) and huSCF (−) which corresponds to the 181 aa vs. 245 aa, respectively. This novel ovine m-SCF (−) has a unique C-terminus with an additional uncharacterized 6 aa residue (176–181, see key to symbols) right after D¹⁷⁵G. Given below the diagram (in 5a, b) are the appropriate topological features (see key to symbols) of human and ovine soluble SCF (+) and membrane-bound SCF (−) with referencce to UniProt ID. P79368 and P21583; (c) Schematic representation of ovine SCF gene transcription and translation in skin (hypothetical view). The corresponding oSCF protein products, s-SCF (+) and m-SCF (−) and their topological characteristics are labeled and highlighted respectively.

https://doi.org/10.1371/journal.pone.0038657.g005

Conservation of the Ovine s-SCF Protein Isoforms

Using the default settings of NCBI, BLASTN and BLASTP search was conducted with ovine s-SCF (+) form of 825 bp CDS and its deduced 274 aa as query sequences, respectively. Multiple sequence alignments (MSA) [64] (Figure S3(a,b,d)) of the nucleotide and the deduced amino acid sequences belonging to different mammalian representatives indicated that the sheep SCF was highly conserved and found to have between 57% and 99% nucleotide similarity and 19% to 99% protein identity (Figure 6(a,d)). The highest identity was with the goat SCF where as the lowest was with gold fish and zebra fish SCF for nucleotide and protein respectively viz. goat (99/99%), cow (97/98%), pig (95/94%), cat (94/90%), panda (93/90%), horse (93/89%), dog (92/88%), human and chimpanzee (91/86%), rabbit (90/84%), marmoset (90/83%), rat (88/82%), mouse (87/80%), zebra finch (74/55%), chicken (73/53%), zebra fish(59/19%) and gold fish (57/26%). The graphical logo representing the conservation of oSCF splice junction (intron-5) with GT repeats, poly(A)₁₁ stretch and the constitutive splice donor (GT) and acceptor (AG) sites are shown in Figure 6(b).

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Figure 6. Graphical representation of evolutionary conservation of sheep SCF isoforms.

(a) Percent of conservation was calculated for sheep, goat, cow, pig, cat, dog, panda, horse, human, chimpanzee, marmoset, mouse, rat, rabbit, chicken, zebra finch and fishes, such as zebra fish, gold fish using the multiple sequence alignment (MSA) tool, ClustalW2 with four different datasets (provided on request). The Circos graphical table view represents the sheep soluble, s-SCF (+) and membrane-bound SCF, m-SCF (−) nucleotide (nt) and protein (aa) as the query sequences (in black dotted left bracket) against 17 other vertebrate species. Four different colour small bars on the query sequences represnts the four different data sets of sheep s-SCF (+) and m-SCF (−) nt/aa sequences. The 15 different colour ribbons passing through each other represent respective vertebrate species and the percent identity is indicated outside as the boundary. The four different colour small bars over the 15 vertebrate species as against 15 different colour small bars above the sheep query sequences represnts the percent identity among each other. The scale over each species (above small bar) represents the total score obtained from the sequence coverage; (b) Graphical logo representing the conservation of oSCF splice junction (intron-5) which was generated by MUSCLE alignment (manually predicted for other species), depicting the GT repeats (black oval dotted lines) proximal to the poly(A)₁₁ stretch (black dotted right brace symbol). The constitutive splice donor (GT) and acceptor (AG) sites are circled by black dotted lines along with one of the proposed usage of alternative/cryptic splice donor site (GT) (see Figure 3f). Numbers below the logo indicate the nucleotide/amino acid position of the MUSCLE aligned sequences; (c) Logo representing the 23 nt conservation of the m-SCF (−) form (novel sequence reported in this study) and its deduced 7 aa new C-terminus is shown; (d) Graphical logo representing the 84 nt conservation of the s-SCF (+) form and its deduced 28 aa proteolytic site is shown. Numbers below the graphical representation of (c), (d) indicate the actual nucleotide/amino acid position. The height of the letters on each logo represents the relative frequency of each nucleotide/amino acid in a given position.

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Similarly, the deduced 181 aa sequence from 546 bp CDS of the ovine m-SCF (−) form shares 49–99% identity with the predicted m-SCF (−) form of the same length of a number of other vertebrate species (Figure 6(a,c); see also Figure S3(a,c)) including avian SCF. The highest identity was with the goat (99%) followed by cow (95%) where as the lowest was noticed with chicken (49%) followed by zebra finch SCF (51%).

Skin Expression of the Two Ovine SCF Splice Variants

Initially, to verify any eventual difference(s) between the expression level of two different splice variants of oSCF (+/−) four sets of primer (summarized in Table S2) were used as described in materials and methods. Three individuals of white, black and brown animals were subjected to a single round RT-PCR amplification. The RT-PCR reactions gave fragments (see Table S2 for details) exhibiting almost the same level of band intensity for both the (+) and (−) form (data not shown). In contrast, Northern blot analysis showed substantial differences in the expression oSCF between (+) and (−) form (Figure 7). At this juncture, we propose that the oSCF gene expression in white, black and brown animals at mRNA transcript level is mediated via an intron-5 AS event (Figure 3(c,d). However, both forms (+/−) are biologically active and reported to have different effects on cells [9]–[11], [20]. The regulation of processing of the proposed secondary proteolytic cleavage site encoded by exon 7, could play a critical role in the function of membrane-associated SCF (−) protein [10].

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Figure 7. Expression of ovine SCF in skin.

Northern blot analysis show ovine SCF (+) and (−) mRNA expression. Ovine 18S rRNA was used as an internal control. Northern blot analysis was carried out with a DIG-labeled cDNA probe for SCF and 18S rRNA (see Table S2) as described in Materials and Methods section. Br, Bl, Wh represents individual of Brown, Black and White merino sheep, respectively.

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SCF UTR Regulatory Motifs that Affect mRNA Stability

The different 5′ and 3′ UTR sequences of sheep SCF(s) were searched against the UTRdbases [86], [92] for the post-transcriptional associated regulatory elements located in the 5′ and 3′ untranslated regions. Among the cis-elements that play a role in translation down-regulation are an upstream open reading frames (uORFs) [106] at nt pos. 80, 193 for the (+) form and at nt pos. 35, 148 for the (−) form; and a polypyrimidine motif, known as terminal oligopyrimidine tract (TOP) [107] at nt pos. 1, 5 located in the 5′ UTR of oSCF (+) form are shown in Figure S4(a₁). The critical regulatory sequences, known as Cytoplasmic Polyadenylation Elements (CPEs), are AU-rich elements (AREs) [108] located in the 3′ UTR near by the canonical nuclear polyadenylation element (AAUAAA), key sequence features controlling mRNA deadenylation and decay. Surprisingly, sheep SCF mRNA has the following single base variant [104] of the type CAUAAA (nt. 1076), AAUGAA (nt. 1080), UUUAAA (nt. 1091), UAUAAA (nt. 1225), AAUAGA (nt. 1441), and AACAAA (nt. 1095, 1183, 1486) for the (+) form (Figure S4(d)) and UAUAAA (nt. 691, 792), AAUAUA (nt. 750, 791) and AGCAAA (nt. 799) for the (−) form (Figure S4(c); see also Figure S4(b)) as its PAS, which are required for proper poly(A) addition. The other regulatory 3′ UTRs found in the oSCF (+) form are Bearded (BRD) Box [109], ‘AGCTTTA’ at nt pos. 1088, 1391 and Musashi binding element (MBE) [110], ‘ATAGT’ at nt. pos. 1422, 1458 (Figure S4(d)).

mRNA Structural Characterization

In addition to the coding region (+/−84 bp proteolytic site), SCF mRNA(s) has four notable features relevant to its secondary structure (Figure 8(a,b)). First, the 5′ UTR is enriched in G+C nucleotides (Figure S4(a₃)), with 64% and 60% or 56% (G+C content) in the 189 nt and 144 nt segment for the (+) and (−) form respectively. Second, the 5′ UTR segment has specific trinucleotide elements (Py-G-C; Figure S4(a₁)), in our case ‘CGC’ at nt pos. 4, 16/18, 33, 36, 68, 100, 154, 176 and and ‘TGC’ at nt pos. 66, 81, 94, 103, 109, 116, 146, 157, 179 that are known to cause DNA polymerase pausing [111]. These trinucleotides (CGC, TGC) which accounts for 9.6% of the ovine s-SCF (+) 5′ UTR segment (189 bp), could attribute to the smaller 5′ RACE cDNA product(s) for example, the one of oSCF isoform-2a (−) (Acc. No. GU386374; Figure S4(a₁)). Third, sheep SCF mRNA contains a frequent hexamer direct repeats (DRs) i.e., ‘CGCTGC’ (1.6%) at nt pos. 100, 154, 176 located in the 5′ UTR of (+) form (also present in (−) form but nt pos. differs; see Figure S4(a₁)). This repeat is highly conserved among mammalian SCF mRNAs (Figure S4(a₂)). Fourth, the 3′ UTR has a DRs containing a consecutive hepatamer ‘GTGGGGG’ at nt pos. 1461, 1468 in the (+) form which is highly conserved only in goat (Figure S4(d)). In contrast, a perfect dinucleotide repeats (GT)₅ at nt pos. 775 is present in between the hepatamer tandem repeats ‘CAAATAT’ at nt pos. 748, 801 in the (−) form, are also highly conserved with goat but varies only in the dinucleotide repeats with ‘AT’ for cow, dog and horse as shown in Figure S4(c). In between this feature, there exists a putative alternative isoform/cryptic splice donor (GT) at nt pos. 117 of 729 bp intron-5 of oSCF (Figure S4(b)) with 92.5% score as predicted by the ASSP [91] classification.

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Figure 8. Ovine SCF RNA fold model explaining the observed results.

(a) Secondary structure predicted for the major 1519 nt s-SCF (+) mRNA. The m-fold circle diagram, generated by minimal free energy (dG = −437.3 kcal/mol) indicate G–C, A–U and G–U base pairs in red, blue and green arc lines, respectively. It also differentiates the 825 nt coding and 505 nt non-coding region fold by the vertical black dotted line which divides the circle diagram. The 189 nt GC-rich segment of 5′ UTR which forms a dense secondary structure and the presumptive splice site of 84 nt proteolytic segment are highlighted and labelled in cyan and dark red dotted line respectively. The start (ATG) and stop (UAA) codons are labeled in bold blue and red letters respectively. The numbers present outside the circle diagram indicates the nucleotide position in base pairs (bp) at every 100 bp intervals; (b) Stemloop secondary structure representation of 1519 nt s-SCF isoform-1 (+), 825 nt m-SCF isoform-2a (−) and the partial image depicting the 5′ UTR segment fold of 725 nt m-SCF isoform-2b (−). The major structural features in illustration (a) and (b) are labeled alike. Except the GC-rich 5′ UTR segments where in I (189 nt), II (144 nt) and III (34 nt) represent the difference in 5′ UTR fold (light blue/cyan dotted arrows pointing to the corresponding dotted oval shape; see also Figure S4(a_1,2,3). Similarly, IV (+84 nt) and V (−84 nt) represents potential fold difference for the proteolytic segments (dotted dark red arrows directd to the corresponding expanded structures).

https://doi.org/10.1371/journal.pone.0038657.g008

MicroRNA Targets: Another Type of cis-acting Regulatory Element

The above described differences between the two ovine splice variants i.e., (+) and (−) in the conservation of non-coding sequences (Figure S4(d)) suggests that the 3′ UTRs, might have a functional role in gene regulation.

A number of potential miRNA target sites are found within the longer ∼4.4 kb 3′ UTR sequence of human SCF (data not shown). However, in sheep, the analyzed miRNA sites that are located in the 505 bp 3′ UTR of the ovine s-SCF (+) form belongs to the miRNA families of miR-27a/b, miR-194, miR-128, miR-370, and two sites for miR-132/212, miR-320/320abcd (Figure 9(a)) where as miR-669f/a/o-3p, miR-466b and miR828b are detected on the shorter 3′ UTR segment (144 bp) of ovine m-SCF (−) form (Figure 9(b)). Interestingly, the 8-mer miRNA (miR-669f) has a high context score (87 percentile) which binds to the 21 nt off 23 nt of the 3′ UTR target of the oSCF (−) form (Figure 9(b)).

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Figure 9. Location of potential miRNA target sites on the 3′ UTR sequences of oSCF (+/−)

. Vertical black bars on the schematic diagram represent miRNA target sites on the 3′ UTR region. Open and dotted boxes represent potential miRNA target sites and sequence conservation, through evolution in sheep, goat, cow, dog, horse and pig. (a) The predicted potential binding site of miR-27a,b on the 3′ UTR of ovine s-SCF (+) and stemloop structure (mfold) of the miR-27a,b is shown. The seed sequences (nt. 2 to nt. 8) of the miR-27a,b is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine s-SCF (+). The nucleotides involved in pairing outside the seed sequence are underlined in black; (b) The predicted potential binding site of miR-669f-3p on the 3′ UTR of ovine m-SCF (−) and stemloop structure (mfold) of the mature miR-669f-3p is shown. The miR-669f-3p target sequence is located on the non-coding intron-5 closest to exon 5. The mature miR-669f-3p is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine m-SCF (−).

https://doi.org/10.1371/journal.pone.0038657.g009

Homology Modeling

The predicted three-dimensional structures of the deduced SCF protein corresponding to 141 aa and 132 aa residues were modelled using the best matched PDB templates with 90–100% identity to the individual chains such as 1EXZ, 2E9W:chainC,D and 1SCF. The structure was predicted as using Modeller 9v2 [54] as described in materials and methods. The quality assessment of the modelled structures were performed at SWISSMODEL Workspace [52].

Topologically, the modelled oSCF structure has a core of four alpha(α)-helices (αA, αB, αC and αD) and two antiparallel beta(β)-strands arranged to form a protomer i.e., β1 between αA and αB and β2 between αC and αD. Apart from this, it consist of three other additional unique conformations i.e., one-turn helix, αB’ between β1 and αB, an hairpin loop between αB and αC at the dimer interface, and an extra one-turn helix, αD’, in the C-terminal extension [112]. This conformation is in accordance with the crystal structure determined for 1EXZ, 1SCF and 2E9W [112]–[114]. The best models were choosen based on the quality assessment reports of ProCheck [115] and Promotif [116]. The calculated Ramachandran plot showed 91–95% of the aa residues lie in the core region for those structures modelled using Modeller 9v2, representing the most favourable combinations of phi-psi values, guiding to the better stereochemical quality of the oSCF protomers while for the one modelled using an automated comparative protein modeling server at SWISS-MODEL, exhibited 70.7% in the core region. Six out of eighteen modelled strcutres were picked and the superimposition of one of the oSCF monomer model to the PDB template 1EXZ:chainB is shown in Figure S5. All these observations suggest correct structure and folding for the modelled putative oSCF.

Molecular Phylogenetic Analyses

The evolutionary divergence of sheep SCF cDNAs and its corresponding protein sequences were studied using other vertebrate sequences from the GenBank, Ensembl and necessary BLAT searches. Except the s-SCF (+) form, the spliceosomal intron junction on the DNA sequences and the m-SCF (−) form were predicted manually in accordance to the ovine SCF sequences.

Five different alignments were constructed for the phylogenetic analysis (data sets provided on request): 1) SCF (+) CDS nucleotide data sets (14 mammals, 2 avian and 2 fish, 822 nt unambiguously aligned characters); and 2) SCF (+) CDS deduced protein sequences (13 mammals, 2 avian and 2 fish, 274 aa unambiguously aligned characters); 3) Predicted SCF (−) CDS nucleotide data sets (12 mammals and 2 avian, 543 nt unambiguously aligned characters) and 4) predicted SCF (−) CDS deduced protein sequences (11 mammals and 2 avian, 181 aa unambiguously aligned characters); (5) Predicted SCF DNA sequences concatenated to the exon 5-Intron(5)-exon 6 (12 mammals and 2 avian, 948 nt unambiguously aligned characters). Unambiguously MUSCLE [47] aligned sequences were confirmed by eye, and unnecessary gaps were excluded from the alignments with GBLOCK program [65] prior to phylogenetic analyses. Phylogenetic relationships were inferred from all five alignments using neighbour-joining (NJ), maximum likelihood (ML) and Bayesian inference (BI) methods as described in materials and methods. The best fit models were scrutinized from 88 nt models [81] and 56 aa models [82] based on the AIC/AICc/BIC/−lnL scores. After the appropriate model selection, the final trees were constructed using the simple p-distance for NJ method, JTT+G, a protein model for ML, BI methods and GTR+G and/or HKY+G for ML, BI as the nucleotide substitutions models. Numbers on the respective nodes denote the supportive bootstrap values of NJ, ML in percentages, and Bayesian posterior probabilities, respectively with the separation of a solidus (/) symbol (Figure S6(a-e)). Apart from the regular GTR+G, HKY+G models, the other useful nucleotide substitution models for our evaluated data sets include TIM3+G, TPM3uf+G, TVM+G, TrN+G and TPM1uf+I+G. All these evaluated models differ in their respective scores by ±5 and produced consistent tree topologies.

All five constructed phylogenetic tree (Figure S6(a-e)), based on oSCF nucleotide and protein sequences (5 different data sets, provided on request) produced similar monophyletic clusters as mammals, avian, and fishes indicating that all the species delineated successfully and was found to be in harmony with the established positioning of these vertebrates. In the tree (Figure S6(c,d,e)) pig-1, pig-2 represents two possible predicted m-SCF amino acid, nucleotide and DNA splice junction sequences, respectively (data sets provided upon request). Note: The s-SCF and m-SCF protein sequence of chimpanzee has 100% identity, hence omitted from the MLA and further tree analyses.

Conclusion

The study that we describe here represents the first attempt to experimentally address the SCF mRNA/cDNA structural coverage in the skin of merino sheep. The analysis of coat color gene(s) structure unique to sheep will extend our understanding of the functional role and regulation of pigmentation genes beyond what was known in mice, humans and other mammals. Here, we have presented evidence for two splice variants of ovine SCF, differing in the cassette exon (CE 6–9/10; skipping of exons 6–9/10) by a premature termination in the non-coding intron 5, which resulted in the presence or absence of a proteolytic site and there by the following transmembrane region and cytoplasmic domain. To our knowledge, this information is previously unreported. Further research is required to determine whether this premature terminated isoform has biological relevance, and whether it leads to the active variant proteins with effects on melanocytic, reproductive or haematological development. The functional role of these two transcripts in ovine skin-specific expression remains unknown. It is important to elucidate which SCF transcript(s), either soluble-SCF (+) or membrane-SCF (−), predominate in the skin which will provide a new insight into an elaborate mechanism involving m-SCF/c-KIT and its counteracting s-SCF/c-KIT signaling that will add to the understanding of the regulation of SCF on hair follicle melanogenesis. In addition, characterization of the SCF promoter(s) is also critical to the design of experiments intended on analysis of the role of various SCF isoforms in vivo using gene targeting techniques. Also, in connection to [33], it would be interesting to determine whether any of the individuals (white, black, and brown) in their families (F₂ generation) have alterations in the SCF gene expression at allele level (QTL/SNPs) or it may have the other alternative splice variant(s)? or lacking any particular reported SCF variants or duplication [147] and/or SCF DNA rearrangement [148]. Future studies exploring other candidate genes are underway especially those involved in the pigmentation regulatory network namely c-KIT and MITF. Altogether, these genes are likely to provide great insight into our understanding of molecular mechanism of the white trait in merino sheep. In this context, further developing ovine chip(s) with key pigmentation associated genetic information such as c-KIT, SCF, MITF, MC1R, ASIP and FGF etc., will open up promising perspectives on using those molecular information in the management of breeding schemes of sheep populations i.e., aiming at Gene Assisted Selection (GAS).