α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism

Table 1

Primers used for PCR, RT-PCR, and qRT-PCR assays to amplify the distinct tvactn gene fragments found in the draft of the T. vaginalis genome sequence, mRNA of each tvactn gene and genes used as controls, complete tvactn3 gene and its three domains for expression, and the different amplicons of the IRE sequences used as RNA probes.

Gene	Location^a (bp)	Primers (5′-3′)
		Forward	Reverse
Tvactn1^b	1512–1648	CCGCTTGCCTTACAGCACTCGG	CCCTTGTTGAAGTGGTCGAGCAT
Tvactn2^b	1551–1676	TACACAGTTCCTCGCCAAGCAG	CCTCTGTTGGGCTGTGGTTGACA
Tvactn3^b	1540–1698	CAGCGCGCTGCCAACGTTGAC	CCAAGGCAAGCTGTGTACATGGCTG
Tvactn4^b	1561–1667	CCTTAAAGCAACAGTTAGCAGCAGC	CCTGTTCGTTTGCTCTTGCAATTAC
Tvactn5^b	1561–1673	CCCCAGGCGAAGAACCAGAAATT	CCGTTTGAGTCCATTGCGACTAACT
𝛽-Tubulin^b	611–722	CATTGATAACGAAGCTCTTTACGAT	GCATGTTGTGCCGGACATAACCAT [25]
Tvactn3^c	1–3390	GGCGGGATCCATGTCTAATAATCGTGGACTTCTAGAC	GGCGGCGGCCGCTTAGGCATAGATAGAATTGAC
Tvactn3 DI^d	1–873	GGCGGGATCCATGTCTAATAATCGTGGACTTCTAGAC	GGCGAAGCTTTCCTGGAACTGTCTGGTCGTAC
Tvactn3 DII^e	688–2400	GGCGGGATCCCACTTCTTCGCTGGCGAGTCA	GGCGGCGGCCGCGACCTTCTCGGCGATGGCAAGAACC
Tvactn3 DIII^f	1984–3390	GGCGGGATCCGACATCACATTCGCCTTCTTGACAC	GGCGGCGGCCGCTTAGGCATAGATAGAATTGAC
Tvpfo a^g	2281–3458	GAAGAGGGCAAGAACTGGGATC	ATCTTCTTGTAGCCCTCGTAA [23]
Tvcp12^h	8–276	GATTTCAAACTTGCTTCCGGCATT	CTTGACTGTTTGGCCCTTGGAAA [25]
IRE-tvcp4^i	−3–28	TAATACGACTCACTATAGGGCACATGTTCGTTCAGGCACCAT	CTTTCTGCTCATGTGCCTGAACGAACATGTG [11]
disrupted IRE-tvcp4^j	12–107	TAATACGACTCACTATAGGGGGCACATGAGCAGAAAGC	GGAGAGCCAAATGCCAAG [11]

aLocation of the region amplified by PCR, RT-PCR, and qRT-PCR of each tvactn gene. ^bPrimers used for RT-PCR and qRT-PCR assays to check Tvactn gene expression under distinct iron concentrations. ^cRestriction sites (underlined) for BamHI and NotI enzymes used to subclone the complete tvactn3 gene from the T. vaginalis CNCD 147 isolate. ^dRestriction sites (underlined) for BamHI and HindIII enzymes used to subclone the Actin-binding domain (DI) of tvactn3 in a bacterial expression system. ^eRestriction sites (underlined) for BamHI and NotI enzymes used to subclone the Spectrin Repeats Domain (DII) of tvactn3 in a bacterial expression system. ^fRestriction sites (underlined) for BamHI and NotI enzymes used to subclone the EF-hand Domain (DIII) of Tvactn3 in a bacterial expression system. ^gPrimers used for RT-PCR assays to check pfo a gene expression under distinct iron concentrations used as an expression control. ^hPrimers used for RT-PCR assays to check tvcp12 gene expression under distinct iron concentrations used as an expression control. ⁱPrimers used for PCR of IRE-tvcp4 sequences. ^jPrimers used for PCR of a deletion  mutant that disrupts the IRE-tvcp4.