Test Excavations at the May Site (CA-SIS-S7) in Seiad Valley, Northwestern California. Joseph L. Chartkoff. Salinas: Coyote Press Archives of California Prehistory No. 17, 1988, vi + 80 pp., 2 maps, 12 figs., 31 tables, $6.20 (paper).

Proximal to transcription start sites, core promoters serve as key regulators of gene expression by sequence-based differential recruitment of transcriptional proteins. Thus, mapping transcription start sites (TSSs) can lead to identification and characterization of core promoter elements, thereby elucidating the relationship between DNA sequence and regulation of gene expression. Using reverse transcription of RNA from active promoters, primer extension can be employed as a quantitative single-step technique for mapping TSSs with high resolution. However, very small amounts of RNA may be challenging to detect with primer extension, and background signals commonly occur due to RNA secondary structures – both issues constrain the utility of primer extension. One possible avenue for addressing these limitations is template-switching: a mechanism that has improved specificity and yield for numerous RNA assays. Thus, we sought to determine whether template-switching can be used to improve primer extension-based analysis of core promoters. In this paper, we outline obstacles and optimizations for implementing template-switching into primer extension. In modifying dNTP concentrations in primer extension to better suit template-switching, we observed highest template-switching efficiency in low overall (<1mM) dNTP concentrations with a relative excess of dCTP; compared to enzymatic alternatives for template-switching, this optimization may be both novel and cost-effective. Furthermore, we found that this template-switching primer extension approach provided both greater sensitivity and dramatically lower background than the traditional primer extension technique. Therefore, we conclude that template-switching effectively enhances primer extension analysis, thus broadening the usefulness of primer extension in studying core promoters.

The tardigrade’s ability to survive in extreme environments intrigues the scientific community. The mechanistic basis for its durable physiology was partially elucidated with the identification of the tardigrade-specific damage suppressor (Dsup) protein, which is a nucleosome-binding factor that protects against DNA damage when expressed in human cells. Because Dsup is only present in certain tardigrades species, however, it is not optimized to function in human cells. Therefore, this study was conducted to determine if a modified Dsup protein containing the nucleosome binding domain (NBD) of the human high mobility group nucleosomal binding domain 2 (HMGN2) protein could improve protection of human cells against reactive oxygen species (ROS), such as hydroxyl radicals, which damage DNA. Two Dsup-HMGN2 hybrid proteins were designed: Dsup containing the entire NBD of human HMGN2 (Dsup-NBD), and Dsup containing only the core NBD region of HMGN2 (Dsup-coreNBD). Dsup wildtype and the two Dsup-HMGN2 hybrids were integrated into the genome of MCF10A cells (human non-tumorigenic epithelial cells), via homology directed repair (HDR) using the CRISPR/Cas9 system. Following exposure to hydrogen peroxide (which generates hydroxyl radicals), the amount of DNA damage in Dsup containing cells was quantified using the alkaline comet assay. Under the conditions of my experiment, I observed significantly more DNA damage in Dsup-expressing cells when compared to the wildtype MCF10A cells lacking Dsup. Despite this conclusion, the optimization of the Dsup protein for DNA damage protection in human cells could have therapeutic potential when used in conjunction with existing technologies such as gene therapy.