Translation of an atypical human cDNA requires fidelity of a purine-pyrimidine repeat region and recoding ☆ Aaron P. Petty a , Charles L. Dick b , J. Suzanne Lindsey a,b,c,d, ⁎ a School of Molecular Biosciences, Washington State University, Pullman, Washington, 99164, USA b Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington, 99164, USA c Center for Reproductive Biology, Washington State University, Pullman, Washington, 99164, USA d Cancer Prevention and Research Center, Washington State University, Pullman, Washington, 99164, USA Received 5 February 2008; accepted 11 February 2008 Received by A.J. van Wijnen Available online 19 February 2008 Abstract Gain or loss of Migration inducting gene-7 (Mig-7) protein expression functional studies suggest it causes aggressive tumor cell invasion and tumor cell vessel-like structure formation. In addition, Mig-7 expression is apparently carcinoma and trophoblast cell-specific. Mig-7 is an example of an atypical gene that is unique in its induction, translation and apparent carcinoma-specific expression. However, studies of this predominantly integral membrane protein are hampered because of the cloning and expression techniques required for detection of Mig-7 protein. Because the encoding region possesses stop codons, repeat sequences and secondary structure, we hypothesized that genetically engineered E. coli are required to maintain the number of purine-pyrimidine repeats and reading frame when producing expression plasmids containing the Mig-7 sequence. Cloning Mig-7 sequence using E. coli genetically engineered to lack recombination and rearrangement capabilities prevented extension of the repeat region. Because of multiple stop codons in the sequence, three different constructs starting from three different reading frame ATG sites were tested for protein production in a human carcinoma cell line. Mig-7 protein of ~ 23 kD is produced from Mig-7 cDNA that contains multiple stop codons downstream from the ATG in a Kozak consensus sequence. In silico analyses imply that multiple Mig-7 mRNA secondary structures may cause frameshifting, read-through, and/or recoding of the multiple stop codons. Experimental results support that one or more of these translational events take place. In this report, we detail requirements for cloning and expression of this novel, atypical, human gene. These techniques can be used to express this unique protein for further studies. © 2008 Elsevier B.V. All rights reserved. Keywords: Mig-7; Z-DNA; Pseudoknot; Polyamines; Recoding 1. Introduction Cloning of genes is routine in molecular biology. Genetic sequences that allow cloning, expression, and detection by standard molecular techniques are most commonly studied. Sequences that are difficult to clone or predicted by current algorithms not to encode a gene remain poorly studied or un- known. One such gene is Mig-7 because its encoding region contains multiple stop codons that should prevent production of the ~23 kD size detected by immunoblot (Crouch et al., 2004; Petty et al., 2007). Available online at www.sciencedirect.com Gene 414 (2008) 49 – 59 www.elsevier.com/locate/gene Abbreviations: Mig-7, Migration inducting gene 7; SURE, Stop Unwanted Rearrangements E. coli; LB, Luria broth; RL95, RL95-2 endometrial carcinoma cells; TBST, Tris borate saline buffer with tween, gt, guanine-thymine; Z-DNA, zig-zag configured DNA. ☆ This research was funded by NIH grant CA93925 (to J.S.L.). ⁎ Corresponding author. Washington State University, College of Pharmacy, Department of Pharmaceutical Sciences, Wegner Hall, Pullman, Washington, 99164-6534, USA. Tel.: +1 509 335 4689; fax: +1 509 335-5902. E-mail address: lindseys@wsu.edu (J.S. Lindsey). 0378-1119/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.02.006