XB-IMG-145576
Xenbase Image ID: 145576
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Supplemental Figure 2: Characterization of DHFR morpholino and . A) DHFR Morpholino sequence. B) Schematic of DHFR exons (E1-E6) to show where the primer sequences and DHFR MO targeted. C) Representative RT-PCR to show that DHFR MO resulted in smaller abnormally spliced DHFR mRNA and reduced levels of normal DHFR. D) Summary of protein sequence comparisons. Full length DHFR protein sequences for selected organisms were retrieved from NCBI as follows: H. sapiens (NP00782.1), M. musculus (NP034179.1), R. norvegicus (NP569084.1), G. gallus (NP001006584.2), X. laevis (NP001088506.1), X. tropicalis (XP004919578.1), and D. rerio (NP571850.1). These full-length protein sequences were compared to humans determine their percent identity using NCBI protein blast (blast.ncbi.nlm.nih.gov). To determine the percent identity for the folate and NADP+ binding sites of DHFR, the residues integral to each site were elucidated using NCBI conserved domain search for each binding site (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Full-length DHFR sequences were aligned using UniProt sequence alignment (http://www.uniprot.org/align/) and the percent identity of all folate binding and NADP+ binding sites compared to H. sapiens was calculated manually. E) The consensus sequence determined from (D) was inputted into the bioinformatics program, Perl, to determine the amount of information at each residue. Shannon’s information equation defines information as H = - , where H is entropy, or the predictability of a sequence of data (1). When applied to the consensus sequence, a high information value represents amino acid residues that are conserved, while low information values represent those that are not. The consensus sequence was superimposed onto the known three-dimensional structure of human DHFR in JMol. Residues were color-coded based on information values in bits. Residues with a bit value of 3.5 or higher were colored red and are the most conserved. Residues with a bit value between 3.0 and 3.4 were colored yellow, those with a bit value between 2.5 and 2.9 were colored green, and those with bit values lower than 2.4 were colored light purple and considered the least conserved. The ligands, NADPH and folate, were also visualized in JMol and colored dark purple and were well conserved. The folate/MTX binding domain (white line) is surrounded almost entirely by red residues. Image published in: Wahl SE et al. (2015) Copyright © 2015. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |