High-throughput gene discovery in the rat

The rat is an important animal model for human diseases and is widely used in physiology. In this article we present a new strategy for gene discovery based on the production of ESTs from serially subtracted and normalized cDNA libraries, and we describe its application for the development of a comprehensive nonredundant collection of rat ESTs. Our new strategy appears to yield substantially more EST clusters per ESTs sequenced than do previous approaches that did not use serial subtraction. However, multiple rounds of library subtraction resulted in high frequencies of otherwise rare internally primed cDNAs, defining the limits of this powerful approach. To date, we have generated >200,000 3′ ESTs from >100 cDNA libraries representing a wide range of tissues and developmental stages of the laboratory rat. Most importantly, we have contributed to ∼50,000 rat UniGene clusters. We have identified, arrayed, and derived 5′ ESTs from >30,000 unique rat cDNA clones. Complete information, including radiation hybrid mapping data, is also maintained locally at http://genome.uiowa.edu/clcg.html. All of the sequences described in this article have been submitted to the dbEST division of the NCBI

Identification of the Retinoic Acid and Thyroid Hormone Receptor-Responsive Element in the Human K14 Keratin Gene

The promoter of human K14 keratin gene, specific for the basal layer of stratified epithelia, is regulated by nuclear receptors for retinoic acid and thyroid hormone. However, the DNA sequences responsible for this regulation have not yet been identified. To identify the retinoic acid-responsive site, we have devised a simple site-specific mutagenesis method and introduced mutations into the K14 keratin gene promoter. These mutations identify the retinoic acid-responsive site. The site consists of a cluster of consensus palindrome half-sites in various orientations. As shown previously, retinoic acid and thyroid hormone receptors can recognize and bind common sequences in regulated genes. Here, we describe mutations that abolish regulation by both receptors. Interestingly, the hormone-dependent and-independent regulatory sites of the thyroid hormone nuclear receptor can be separated. Clusters of half-sites that share structural organization with the K14 regulatory site were found in the K5 and K10 keratin gene promoters. Similar clusters may be responsible for retinoic acid-mediated transcription regulation in epidermis

Cells Expressing Murine RAD52 Splice Variants Favor Sister Chromatid Repair

The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired