Applying gene expression, proteomics and single-nucleotide polymorphism analysis for complex trait gene identification.

Previous quantitative trait locus (QTL) analysis of an intercross involving the inbred mouse strains NZB/BlNJ and SM/J revealed QTL for a variety of complex traits. Many QTL have large intervals containing hundreds of genes, and methods are needed to rapidly sort through these genes for probable candidates. We chose nine QTL: the three most significant for high-density lipoprotein (HDL) cholesterol, gallstone formation, and obesity. We searched for candidate genes using three different approaches: mRNA microarray gene expression technology to assess >45,000 transcripts, publicly available SNPs to locate genes that are not identical by descent and that contain nonsynonymous coding differences, and a mass-spectrometry-based proteomics technology to interrogate nearly 1000 proteins for differential expression in the liver of the two parental inbred strains. This systematic approach reduced the number of candidate genes within each QTL from hundreds to a manageable list. Each of the three approaches selected candidates that the other two approaches missed. For example, candidate genes such as Apoa2 and Acads had differential protein levels although the mRNA levels were similar. We conclude that all three approaches are important and that focusing on a single approach such as mRNA expression may fail to identify a QTL gene.

Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity.

Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.

Validation of DNA sequences using mass spectrometry coupled with nucleoside mass tagging.

We present a mass spectrometry (MS)-based nucleoside-specific mass-tagging method to validate genomic DNA sequences containing ambiguities not resolved by gel electrophoresis. Selected types of (13)C/(15)N-labeled dNTPs are used in PCR amplification of target regions followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-MS analysis. From the mass difference between the PCR products generated with unlabeled nucleosides and products containing (13)C/(15)N-labeled nucleosides, we determined the base composition of the genomic regions of interest. Two approaches were used to verify the target regions: The first approach used nucleosides partially enriched with stable isotopes to identify a single uncalled base in a gel electrophoresis-sequenced region. The second approach used mass tags with 100% heavy nucleosides to examine a GC-rich region of a polycytidine string with an unknown number of cytidines. By use of selected (13)C/(15)N-labeled dNTPs (dCTPs) in PCR amplification of the target region in tandem with MALDI-TOF-MS, we determined precisely that this string contains 11 cytidines. Both approaches show the ability of our MS-based mass-tagging strategy to solve critical questions of sequence identities that might be essential in determining the proper reading frames of the targeted regions.