Analysis of the DBR-1 Gene
Analysis of the RBM22 Protein Association with Gene Expression through Affinity Tagging
Lucas Akin
Stream Faculty: Scott Stevens
Research Professor: Albert MacKrell
University of Texas at Austin
School of Biological Sciences
Abstract
Affinity tagging of proteins is an important method for biochemical analyses [4] and has proven effective in further expanding our knowledge of their specific functions. A group of interactive proteins expressed by bacteriophage ʎ known as exo, bet, and gam have shown a unique ability to facilitate homologous recombination [4,5,6, and 7]. We describe the techniques behind the recombineering process that enables us to insert a modified affinity tag with neomycin resistance known as “TAP-neo” in between the Z1 and Z2 fragments of the RBM22 gene. Similar research has shown evidence of successful recombination events [3,5 and 6] that have ultimately led to purification procedures of the affinity tagged fusion protein complexes which permit mass spectrophotometric analysis [1,2 4,5,6,7 and 8]. Optimization of the earlier steps within this experiment that serve as preliminary procedures needed to yield a purified plasmid product from the pGEM-T Easy (Promega) vector are described in detail. Future testing is needed before beginning recombineering, selection of homologous recombination and purification of the TAP-tagged RBM22 protein product.
Introduction
The RNA-binding motif protein 22 (RBM22) has known interactive functions involved in the early stages of pre-mRNA splicing activity [2,3] as well as nuclear translocation of certain calcium-binding proteins [2]. It contains a conserved RNA-binding domain, a zinc finger [2,3] uniquely downstream of a C-terminus rich in the amino acids Glycine and Proline [2]. Found in both the nucleus and cytoplasm of eukaryotes and yeast [2,3], RBM22 binds directly to the internal stem-loop (ISL) domain of theU6 snRNA and to the pre-mRNA intron near the 5 ' splice site [3].
Lucas Akin
Stream Faculty: Scott Stevens
Research Professor: Albert MacKrell
University of Texas at Austin
School of Biological Sciences
Abstract
Affinity tagging of proteins is an important method for biochemical analyses [4] and has proven effective in further expanding our knowledge of their specific functions. A group of interactive proteins expressed by bacteriophage ʎ known as exo, bet, and gam have shown a unique ability to facilitate homologous recombination [4,5,6, and 7]. We describe the techniques behind the recombineering process that enables us to insert a modified affinity tag with neomycin resistance known as “TAP-neo” in between the Z1 and Z2 fragments of the RBM22 gene. Similar research has shown evidence of successful recombination events [3,5 and 6] that have ultimately led to purification procedures of the affinity tagged fusion protein complexes which permit mass spectrophotometric analysis [1,2 4,5,6,7 and 8]. Optimization of the earlier steps within this experiment that serve as preliminary procedures needed to yield a purified plasmid product from the pGEM-T Easy (Promega) vector are described in detail. Future testing is needed before beginning recombineering, selection of homologous recombination and purification of the TAP-tagged RBM22 protein product.
Introduction
The RNA-binding motif protein 22 (RBM22) has known interactive functions involved in the early stages of pre-mRNA splicing activity [2,3] as well as nuclear translocation of certain calcium-binding proteins [2]. It contains a conserved RNA-binding domain, a zinc finger [2,3] uniquely downstream of a C-terminus rich in the amino acids Glycine and Proline [2]. Found in both the nucleus and cytoplasm of eukaryotes and yeast [2,3], RBM22 binds directly to the internal stem-loop (ISL) domain of theU6 snRNA and to the pre-mRNA intron near the 5 ' splice site [3].
References: 1. Rea M. Lardelli, James X. Thompson, John R. Yates III, et al. (2010) Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing. RNA Society. 516-528 2 3. F. He, C.-T. Wang, L.-T. Gou. (2009) RNA-binding motif protein RBM22 is required for normal development of zebrafish embryos. Journal of Genetics and Molecular Research. 1466-1473 4 5. Dewang Zhou, Jin-Xiang Ren, Thomas M. Ryan, N. Patrick Higgins and Tim M. Townes. (2004) Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts. Nucleic Acids Research. Vol. 32, No. 16 6 7. J. A. Mosberg, M. J. Lajoie and G. M. Church. (2010) Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate. Genetics Society of America. Vol. 186, No. 191-199 8