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Characterization of the bovine pregnancy-associated glycoprotein gene family--analysis of gene sequences, regulatory regions within the promoter and expression of selected genes.

Telugu BP, Walker AM, Green JA - BMC Genomics (2009)

Bottom Line: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order.However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern boPAG grouping, but not the ancient boPAGs.These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA. telugub@missouri.edu

ABSTRACT

Background: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order. In cattle, the PAG gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1) we performed an in silico analysis of the bovine genome to further characterize the PAG gene family, 2) we scrutinized proximal promoter sequences of the PAG genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected PAGs during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, bovine (bo) PAG-2.

Results: From our analysis of the bovine genome, we identified 18 distinct PAG genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all boPAGs. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern boPAG grouping, but not the ancient boPAGs. We gathered evidence by means of Q-PCR and screening of EST databases to show that boPAG-2 is the most abundant of all boPAG transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the boPAG-2 gene.

Conclusion: PAGs represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed differences in spatial and temporal expression. We also discovered that boPAG-2 is the most abundant of all boPAG transcripts and provided evidence for the role of ETS and DDVL TFs in its regulation. These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.

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The ratio of p-distance (p-dist) of the promoter regions versus predicted nucleotide mutation rate [calculated as dS (proportion of synonymous substitutions per synonymous site in the exons)] in pairwise comparisons for each PAG gene represented in the genome build. A. Comparisons with the proximal 1000 bp of the promoter region. B. Comparisons with the proximal 500 bp of the promoter region. The p-distance of the promoters was shown on the Y-axis and the dS of their protein coding regions were displayed on the X-axis. The unique marks of a particular color and shape in the figure represent the pairwise comparisons of boPAG against each of the other PAGs included in the analysis. The listing of PAG genes and their indicators are shown in the legend.
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Figure 3: The ratio of p-distance (p-dist) of the promoter regions versus predicted nucleotide mutation rate [calculated as dS (proportion of synonymous substitutions per synonymous site in the exons)] in pairwise comparisons for each PAG gene represented in the genome build. A. Comparisons with the proximal 1000 bp of the promoter region. B. Comparisons with the proximal 500 bp of the promoter region. The p-distance of the promoters was shown on the Y-axis and the dS of their protein coding regions were displayed on the X-axis. The unique marks of a particular color and shape in the figure represent the pairwise comparisons of boPAG against each of the other PAGs included in the analysis. The listing of PAG genes and their indicators are shown in the legend.

Mentions: The analysis was performed with two variable lengths of promoter sequence. When the p-distance v. the ORF for the proximal 1000 bp was mapped, all of the boPAGs were undergoing neutral to purifying selection (Figure 3A and 3B), with the exception of boPAG-10 and -6, which had ratios of more than one (Figure 3A). These promoters seemed to have accumulated more mutations than would have been predicted by molecular clocks. The analysis, when confined to the first 500 bp, generated similar results except that both boPAG-6 and -10 showed a ratio close to neutrality (Figure 3B). Overall, the boPAG promoters are being conserved, particularly in the first 500 bp upstream of the TSS (Figure 3B) implying that critical regulatory elements responsible for trophoblast expression may be positioned within this region.


Characterization of the bovine pregnancy-associated glycoprotein gene family--analysis of gene sequences, regulatory regions within the promoter and expression of selected genes.

Telugu BP, Walker AM, Green JA - BMC Genomics (2009)

The ratio of p-distance (p-dist) of the promoter regions versus predicted nucleotide mutation rate [calculated as dS (proportion of synonymous substitutions per synonymous site in the exons)] in pairwise comparisons for each PAG gene represented in the genome build. A. Comparisons with the proximal 1000 bp of the promoter region. B. Comparisons with the proximal 500 bp of the promoter region. The p-distance of the promoters was shown on the Y-axis and the dS of their protein coding regions were displayed on the X-axis. The unique marks of a particular color and shape in the figure represent the pairwise comparisons of boPAG against each of the other PAGs included in the analysis. The listing of PAG genes and their indicators are shown in the legend.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682831&req=5

Figure 3: The ratio of p-distance (p-dist) of the promoter regions versus predicted nucleotide mutation rate [calculated as dS (proportion of synonymous substitutions per synonymous site in the exons)] in pairwise comparisons for each PAG gene represented in the genome build. A. Comparisons with the proximal 1000 bp of the promoter region. B. Comparisons with the proximal 500 bp of the promoter region. The p-distance of the promoters was shown on the Y-axis and the dS of their protein coding regions were displayed on the X-axis. The unique marks of a particular color and shape in the figure represent the pairwise comparisons of boPAG against each of the other PAGs included in the analysis. The listing of PAG genes and their indicators are shown in the legend.
Mentions: The analysis was performed with two variable lengths of promoter sequence. When the p-distance v. the ORF for the proximal 1000 bp was mapped, all of the boPAGs were undergoing neutral to purifying selection (Figure 3A and 3B), with the exception of boPAG-10 and -6, which had ratios of more than one (Figure 3A). These promoters seemed to have accumulated more mutations than would have been predicted by molecular clocks. The analysis, when confined to the first 500 bp, generated similar results except that both boPAG-6 and -10 showed a ratio close to neutrality (Figure 3B). Overall, the boPAG promoters are being conserved, particularly in the first 500 bp upstream of the TSS (Figure 3B) implying that critical regulatory elements responsible for trophoblast expression may be positioned within this region.

Bottom Line: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order.However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern boPAG grouping, but not the ancient boPAGs.These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA. telugub@missouri.edu

ABSTRACT

Background: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order. In cattle, the PAG gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1) we performed an in silico analysis of the bovine genome to further characterize the PAG gene family, 2) we scrutinized proximal promoter sequences of the PAG genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected PAGs during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, bovine (bo) PAG-2.

Results: From our analysis of the bovine genome, we identified 18 distinct PAG genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all boPAGs. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern boPAG grouping, but not the ancient boPAGs. We gathered evidence by means of Q-PCR and screening of EST databases to show that boPAG-2 is the most abundant of all boPAG transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the boPAG-2 gene.

Conclusion: PAGs represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed differences in spatial and temporal expression. We also discovered that boPAG-2 is the most abundant of all boPAG transcripts and provided evidence for the role of ETS and DDVL TFs in its regulation. These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.

Show MeSH