Supplementary MaterialsS1 Table: Sequence of primers targeting PAG genes. PCR product of size ~1.2 kb was observed at 45 days (lane 1), 75 days (lane 2) and 90 days (lane 3) pregnancy. Lane 4 represents the DNA ladder.(DOCX) pone.0206143.s004.docx (224K) GUID:?C1E606E3-21D7-434C-A965-40FAE32D1D58 S2 Fig: Percent identity matrices of variants belonging to (A) BuPAG 2 (B) BuPAG 7 (C) BuPAG 8 (D) BuPAG Odanacatib pontent inhibitor 16 and (E) BuPAG 18.(DOCX) pone.0206143.s005.docx (2.6M) GUID:?07FAC119-70D5-4396-A36A-A02A78CD9178 S3 Fig: Evolutionary relationships among different isoforms of BuPAGs and their variants: The tree was created from the deduced amino acid sequences by the Neighbor Joining method in the MEGA 4.0 program. The tree was drawn to scale, and the numbers on the branches represent the confidence levels obtained from the bootstrap analysis (1000 replicates).(DOCX) pone.0206143.s006.docx (198K) GUID:?3F5A8A1E-5E71-473D-9206-EEB6303C438C Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Pregnancy-associated glycoproteins (PAGs) are expressed during pregnancy by the trophoectodermal cells of fetus. Presence of PAGs in dam’s circulation has been widely used in pregnancy diagnosis. The present study reports the identification and characterization of different PAG isoforms in buffalo PR22 during early stages of pregnancy. The mRNAs isolated from fetal cotyledons (Pregnancy stages: 45, 75 and 90 days) were successfully cloned in Odanacatib pontent inhibitor pJET1.2 vector and transformed in were collected from local Odanacatib pontent inhibitor slaughter house around Karnal, India. Animals Odanacatib pontent inhibitor were not slaughtered specifically for sample collection and hence ethical clearance was not required to collect the samples. Fetuses were retrieved and the age of pregnancy was determined by measuring their crown to rump (C-R) length [44]. Individual cotyledons were collected, washed with DEPC-treated water (0.1%) and stored in RNA later solution (Sigma Chem. Co.) at -20C till further use. All samples were mainly categorized into three groups i.e. 45 days, 75 days and 90 days of pregnancy. Each group contained samples from 4 different fetuses. Total RNA was extracted from the cotyledonary tissue by using TRIzol reagent (Invitrogen, USA) as per manufacturer’s instructions. The possible genomic DNA contamination in prepared RNA samples was removed by using DNA free kit (Ambion, USA). The total RNA extracted was quantified by measuring the ratio of absorbance at 260/280 nm wavelength using the Infinite 200 PRO NanoQuant system (Tecan, Austria). The purity and integrity of prepared RNA samples were further verified by 1.2% agarose gel electrophoresis. Twenty two PAG isoform sequences of were retrieved from GenBank nucleotide database (GenBank: L27833.1, NM_176614, XM_615231, NM_176615.2, NM_176616, NM_176617.2, BC133469.1, NM_176619.2, NM_176620.2) and were aligned using Clustal W (1.82) for primer designing. A total of Odanacatib pontent inhibitor 6 sets of primers were designed for full length amplification of by analyzing the conserved sequences in the upstream and downstream regions of ORF, using Primer3 software hosted at NCBI (S1 Table). For the amplification of most of the isoforms, one set of primers i.e. BoPAGF(conserved) and BoPAGRcommon was designed by analyzing the most conserved regions of isoforms of species. For the amplification of other isoforms i.e. 1, 3, 4 and 5; individual forward primers namely, and genes for and available at NCBI GenBank database using the program pBLAST. The sequence nomenclatures were decided on the basis of maximum similarity of the sequences with and available in the database. For all the sequences matching maximally with a particular reported isoform, multiple alignments were done using the MegAlign module of DNASTAR software to check whether they are the same sequences or they are the variants of a particular isoform. The identified isoforms were classified into three separate groups of pregnancy namely, 45 days, 75 days and 90 days. The relative abundance of each BuPAG isoform at protein level was calculated as the percentage of total screened colonies in each group and the trend analysis was performed to analyze how the expression of identified BuPAG isoforms vary across the selected stages of pregnancy. The signal peptide prediction for all the identified isoforms was performed using online SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/) based on neural network trained on eukaryotes [45]. Physico-chemical properties were analyzed using Protparam server at ExPASy. The conserved domains were identified using PROSITE database at ExPASy [46]. The multiple alignments and percent identities among the identified BuPAGs and other reported PAGs in cow and buffalo were determined using Megalign module of DNASTAR software. Phylogenetic analysis To study the evolutionary relationship of BuPAGs, the amino acid sequences of different bovine PAG isoforms (boPAG), PAG-like molecules and other mammalian aspartic proteinases were downloaded from NCBI with following accession numbers:boPAG 1 (AAB35845.1), boPAG 2 (NP_788787.1), boPAG 4 (NP_788788.1), boPAG 5.