Figure 1.

Expression profiling and genomic knockout of MMA1. (A) The expression profile of MMA1 (red trace) is highly similar to that of SOR4 (blue trace), which encodes a receptor required for mucocyst biogenesis. The profiles of transcript abundance under a variety of culture conditions, derived via hybridization of stage-specific cDNAs to whole genome microarrays, are from the Tetrahymena Functional Genomics Database (http://tfgd.ihb.ac.cn/). In the plots shown, each trace was normalized to that gene’s maximum expression level. The culture conditions sampled at successive time points represent growing (L-l, L-m, and L-h), starved (S-0, S-3, S-6, S-9, S-12, S-15, and S-24), and conjugating (C-0, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16, and C-18) cultures. For details on the sampling conditions, see Miao et al. (2009). (B) Schematic of MMA1 Macronuclear gene knockout construct. Replacement of the Macronuclear MMA1 gene by the Neo4 drug resistance cassette was targeted by homologous recombination. (C) Confirmation of MMA1 Macronuclear knockout by RT-PCR. RNA was extracted from wild type, UC620, and ∆mma1, converted to cDNA, and PCR amplified using MMA1-specific primers listed in Table S1. A 1% ethidium bromide-stained agarose gel is shown. The MMA1 product (indicated by arrow), which was confirmed by sequencing, was present in wild type and UC620 samples but absent in ∆mma1. The larger amplicon present in all lanes corresponds to MMA1 with the intron still present, as determined by sequencing, and thus likely reflects amplification of a genomic (Micronuclear) DNA contaminant. Parallel amplification of the α−tubulin gene from all samples was used to control for sample loading.