Abstract
Understanding the transcriptome, defined as the complete transcriptional component of the genome, is far more complex than originally considered. Even with the near fully resolved human and mouse genomes, for which extensive databases of transcribed sequence data (e.g., expressed sequence tags) are available, it is presently not possible to experimentally recover or computationally predict the full range of transcription products that derive from multiexon genes. Many genes are tightly regulated, which could include alternative processing of RNA, and lead to significant underrepresentation of many transcripts. A multitude of factors in addition to cell lineage- and developmental stage-specific expression as well as shortcomings in computational methods result in a less than complete understanding of transcriptional complexity. Here, we describe an approach to predict and evaluate a more complete repertoire of transcriptional products that derive from specific genetic loci with attention toward analysis of immune receptor genes. This approach is particularly useful in identifying gene products, including alternative splice forms, that originate from complex multigene families.
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Salzberg, S., and Yorke, J. (2005) Beware of mis-assembled genomes. Bioinformatics 21, 4320–1.
Amemiya, C. T., Ota, T., and Litman, G. W. (1996) in “Nonmammalian Genomic Analysis: A Practical Guide” (Birren, B., and Lai, E., Eds.), Academic Press, Inc., San Diego.
Graham, F., Smiley, J., Russell, W., and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36, 59–74.
Shaw, G., Morse, S., Ararat, M., and Graham, F. (2002) Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK293T cells. FASEB J 16, 869–71.
Zhang, Y., and Frohman, M. (1997) Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs. Methods Mol Biol 69, 61–87.
Amemiya, C., Prohaska, S., Hill-Force, A., Cook, A., Wasserscheid, J., Ferrier, D., Pascual-Anaya, J., Garcia-Fernàndez, J., Dewar, K., and Stadler, P. (2008) The amphioxus Hox cluster: characterization, comparative genomics, and evolution. J Exp Zool B Mol Dev Evol 310, 465–77.
Amemiya, C., and Zon, L. (1999) Generation of a zebrafish P1 artificial chromosome library. Genomics 58, 211–3.
Kim, S., Horrigan, S., Altenhofen, J., Arbieva, Z., Hoffman, R., and Westbrook, C. (1998) Modification of bacterial artificial chromosome clones using Cre recombinase: introduction of selectable markers for expression in eukaryotic cells. Genome Res 8, 404–12.
Heintz, N. (2001) BAC to the future: the use of bac transgenic mice for neuroscience research. Nat Rev Neurosci 2, 861–70.
Giraldo, P., and Montoliu, L. (2001) Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res 10, 83–103.
Al-Hasani, K., Simpfendorfer, K., Wardan, H., Vadolas, J., Zaibak, F., Villain, R., and Ioannou, P. (2003) Development of a novel bacterial artificial chromosome cloning system for functional studies. Plasmid 49, 184–7.
Wang, Z., Longacre, A., and Engler, P. (2004) Retrofitting BACs with a selectable marker for transfection. Methods Mol Biol 256, 69–76.
Sparwasser, T., and Eberl, G. (2007) BAC to immunology–bacterial artificial chromosome-mediated transgenesis for targeting of immune cells. Immunology 121, 308–13.
Dishaw, L., Mueller, M., Gwatney, N., Cannon, J., Haire, R., Litman, R., Amemiya, C., Ota, T., Rowen, L., Glusman, G., and Litman, G. (2008) Genomic complexity of the variable region-containing chitin-binding proteins in amphioxus. BMC Genet 9, 78.
Yoder, J., Litman, R., Mueller, M., Desai, S., Dobrinski, K., Montgomery, J., Buzzeo, M., Ota, T., Amemiya, C., Trede, N., Wei, S., Djeu, J., Humphray, S., Jekosch, K., Hernandez Prada, J., Ostrov, D., and Litman, G. (2004) Resolution of the novel immune-type receptor gene cluster in zebrafish. Proc Natl Acad Sci U S A 101, 15706–11.
Tsaftaris, A., Pasentzis, K., and Argiriou, A. (2010) Rolling circle amplification of genomic templates for inverse PCR (RCA-GIP): a method for 5’- and 3’-genome walking without anchoring. Biotechnol Lett 32, 157–61.
Tsuchiya, T., Kameya, N., and Nakamura, I. (2009) Straight walk: a modified method of ligation-mediated genome walking for plant species with large genomes. Anal Biochem. 388, 150–60.
Parimoo, S., Patanjali, S., Shukla, H., Chaplin, D., and Weissman, S. (1991) cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments. Proc Natl Acad Sci U S A 88, 9623–7.
Lovett, M., Kere, J., and Hinton, L. (1991) Direct selection: a method for the isolation of cDNAs encoded by large genomic regions. Proc Natl Acad Sci U S A 88, 9628–32.
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Dishaw, L.J., Mueller, M.G., Haire, R.N., Litman, G.W. (2011). Transfection-Based Genomic Readout for Identifying Rare Transcriptional Splice Variants. In: Rast, J., Booth, J. (eds) Immune Receptors. Methods in Molecular Biology, vol 748. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-139-0_17
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DOI: https://doi.org/10.1007/978-1-61779-139-0_17
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