No abstract

Transcription factor IIIC (TFIIIC) (or tau) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, tau91, present in affinity-purified TFIIIC. Tau91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of tau91 was established by the following biochemical and genetic evidence. Antibodies to tau91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to tau91; and a point mutation in TFC6 (tau91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349E mutant affected in the B block binding subunit (tau138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that tau91 cooperates with tau138 for DNA binding. Recombinant tau91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

Fibres of cotton (Gossypium hirsutum L.) are single elongated epidermal cells that start to develop on the outer surface of cotton ovules on the day of anthesis. Little is known about the control of fibre initiation and development. As a first step towards discovering important genes involved in fibre initiation and development using a genomics approach, we report technical advances aimed at reducing redundancy and increasing coverage for anonymous cDNA microarrays in this study. Cotton ovule cDNA libraries (both normalised and un-normalised) from around the time of fibre initial formation have been prepared and partially characterised by sequencing. Re-association-based normalisation partially reduced library redundancy and increased representation of novel sequences. However, another library generated from in vitro cultured cotton ovules treated with the protein synthesis inhibitor, cycloheximide, showed a significantly altered gene representation including a greater proportion of protein phosphorylation genes, transport genes and transcription factors and a much reduced proportion of protein synthesis genes than were identified in the conventional types of libraries. Over 10,000 expressed sequence tag (EST) clones randomly selected from the three libraries were printed on microarray slides and used to assess gene expression in tissue cultured ovules with and without cycloheximide treatment. The microarray results showed that cycloheximide had a dramatic effect in modifying the pattern of the gene expression in cultured ovules, affecting the same types of genes identified in the preliminary analysis on relative EST abundance in the different ovule cDNA libraries. Cycloheximide clearly provided a simple and useful method for enriching novel gene sequences for genomic studies.