Friday, November 16, 2012

METHODS AND RESULTS

        Allikmets, Gerrard, Hutchinson, and Dean (1996) characterized a large number of human ABC transporter genes by searching the human EST database using sequences derived from ABC (Walker A, ABC signature, and Walker B) of MDR1, as well as the entire sequence of cystic fibrosis transmembrane conductane regulator (CFTR). Because many of the ESTs are derived from brain libraries, the data suggested that many of these ABC transporters were expressed in brain. Because the number of ESTs deposited in public databases has increased substantially in the past 4 years, we recently repeated this analysis in order to ensure that the full complement of brain-expressed ABC transporters was represented in the database. Moreover, to increase the power of our search, we developed consensus amino acid sequences for the Walker A motif, as well as the ABC signature and Walker B motifs, by comparing the sequences of the following human ABC transporters.  The consensus sequences that emerged from this analysis were (G(X)2G(X)GK(X)T(X)4L(X)2L(X)2PT(X)3G for the Walker A motif, and LSGG(X)4L(X)2A(X)AL(X)3PKV(X)2LDE(X)TS(X) for the ABC signature and Walker B motifs.

       The BLAST programs (Altschul, Gish, Miller, Myers, & Lipman, 1990) were used to search the human database EST using these consensus sequences, as well as the complete amino acid sequences of the human ABC-transporter proteins identified above. Human clones with the highest scores were retrieved and primers were designed using the program Mac Vector (Oxford Molecular, UK). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using brain fetal and adult total RNA (Invitrogen) in order to confirm human brain expression, and PCR products were sequenced to authenticate the ESTs.

       Based on both the cDNA library source used to generate these ESTs, as well as the expression profile of RNA as seen in Northern blots (Allikmets et al. 1996, and unpublished data), we identified the 14 ABC transporters listed in Table 10.1 as being brain-expressed.

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