4 kb); I-BET-762 chemical structure EF650850 (MTT1-BS07 2.4 kb); EF650851 (MTT1-BS07 2.7 kb); EF650852 (MTT1-A15 2.4 kb) and EF650853 (MTT1-WS 2.7 kb). Previously deposited sequences
are available under accession numbers DQ010168–DQ010174. Sequences were aligned using clustalw (Thompson et al., 1994). prosite was used to find motifs and membrane-spanning domains in the purported proteins (http://www.expasy.org/prosite/). Binding sites in the promoters were analysed using siteseer (Boardman et al., 2003). We have reported previously that antimycin A strongly inhibits the growth of lager strain A15 on a solid medium with maltotriose, but has less effect on the growth of lager strain WS34/70 (Dietvorst et al., 2005). As shown in Fig. 1, lager strain BS07 was also inhibited in its growth on maltotriose
in the presence of antimycin A, but to a smaller extent than lager strain A15. A fourth lager yeast strain, BS01, shows a similar growth profile as strain WS34/70. For growth on maltose as a carbon source, the effect of antimycin A was less and about the same for all four strains (Fig. 1). To investigate the presence of MTT1-like and/or selleck kinase inhibitor MAL31-like genes in the lager yeast strains A15, WS34/70, BS01 and BS07, PCRs were performed using specific primer combinations MAL31-fw – MAL31-rv and Mty1-fw – Mty1-rv, respectively (Table 1 and Fig. 2). These primers discriminate between MTT1- and MAL31-like genes. Using these primers, we showed that all four lager strains contain both MAL31 and MTT1 genes (data not shown). To isolate MAL31 Cell press and MTT1 genes from the four lager yeast strains, independent PCRs were performed using the universal primers ‘MAL31Xba’and ‘MAL31BamH’. This PCR amplifies the sequences between 542 or 836 bp upstream and 26 bp downstream of the open reading frame (ORFs) and yielded both 2.4- and 2.7-kb products as reported previously for strains A15 and
WS34/70 (Dietvorst et al., 2005). The PCR products were inserted into the pCR-TOPO vector and independent clones were isolated and characterized by PCR with the MTT1-specific pimers Mty1-fw and Mty1-rv and the MAL31-specific primers Mal31-fw and Mal31-rv (Table 1 and Fig. 2). From strains WS34/70 and BS07, both 2.4- and 2.7-kb versions of the MAL31 and MTT1 genes were isolated, whereas the 2.7-kb version of MTT1 was not found in strains A15 and BS01 (Table 2). To further study the role of these genes in maltotriose metabolism, the various MAL31 and MTT1 genes were recloned into the multicopy vector pRUL409(KanMX). A15 transformants containing these constructs were tested for their ability to start growing rapidly on maltotriose in the presence of antimycin A. For each strain, at least two independent isolates of both the 2.4- and the 2.7-kb versions of both the MAL31 and the MTT1 genes were tested in this manner, except for the 2.7-kb MTT1 versions from strains BS01 and A15, which were not found. Transformants carrying the 2.