Possible Identification of Schizosaccharomyces pombe gene(s) Encoding POU Domain Transcription Factors

Abstract

Eukaryotic RNA polymerase II transcription is characterized by a complex array of various transcription factors which recognize and bind to specific DNA sequence elements in promoters for accurate initiation of transcription and in enhancers for increased transcriptional rates. A novel family of transcription factors which share binding site specificities and a conserved domain is called the POU domain family. The goal of this study was to attempt to identify gene(s) in the fission yeast Schizosaccharomyces pombe that encode POU domain transcription factors. Two methodologies were employed. Southern blotting of yeast chromosomal DNA with probes derived from the cloned mammalian gene encoding Oct-1 (which is a POU domain-containing factor) were done in an attempt to find homologous yeast nucleotide sequences. Results from these Southern blots suggested a weak cross hybridization of yeast DNA when the mammalian Oct-1 gene was used as a probe. Another attempt to find homologous yeast POU domain encoding gene(s) was based on polymerase chain reaction (PCR) of S. pombe cDNAs using a set of synthesized degenerate oligonucleotides corresponding to seven highly conserved amino acids in the POU domain. The other primer was the universal (-20) primer. The PCR products were cloned into a plasmid vector, and double stranded dideoxy DNA sequencing has revealed the orientation that a possible POU domain encoding gene has (3' to the insertion site). Currently, sequencing reactions using the forward (-40) primer are being done to sequence through this stretch of DNA to determine if it encodes a POU domain (by looking at the predicted amino acid sequences).