To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.

SummaryA large and diverse set of proteins, including CST complex, nonsense mediated decay (NMD), and DNA damage response (DDR) proteins, play important roles at the telomere in mammals and yeast. Here, we report that NMD, like the DDR, affects single-stranded DNA (ssDNA) production at uncapped telomeres. Remarkably, we find that the requirement for Cdc13, one of the components of CST, can be efficiently bypassed when aspects of DDR and NMD pathways are inactivated. However, identical genetic interventions do not bypass the need for Stn1 and Ten1, the partners of Cdc13. We show that disabling NMD alters the stoichiometry of CST components at telomeres and permits Stn1 to bind telomeres in the absence of Cdc13. Our data support a model that Stn1 and Ten1 can function in a Cdc13-independent manner and have implications for the function of CST components across eukaryotes.

A simple nonsurgical technique of obtaining fat samples by aspiration from the gluteal prominence was developed by Hirsh in 1960 and has been in use in our Nutrition Clinic at the Mount Sinai Hospital for several years. We have modified it for field use and the analysis of fat-soluble hydrocarbon residues. All the materials which will contain the fat sample to be analyzed are washed with acetone and pesticide residue-free hexane, and a 15 gauge needle and 33 cc syringe are sterilized. Aspiration of fat from the lateral gluteal prominence is accomplished under local xylocaine anesthetic. The anesthetic also serves as the vehicle into which the fat is broken by the shearing action of the 15 gauge needle. Fat particles are sucked into the syringe by a constant vacuum kept on the syringe during lateral movement of the needle under (and parallel to) the skin within the gluteal fat pad; 200--500 ml of fat can be obtained for hydrocarbon residue analysis. The only complications have been some mild hematomas at the site of the aspiration. The method avoids surgical biopsy and sutures and takes about 7--8 min.ImagesFIGURE 1. AFIGURE 1. BFIGURE 2.FIGURE 3.

Single-stranded DNA (ssDNA) intermediates play an important role in processes such as DNA replication and homologous recombination, DNA damage responses, and DNA repair. Using quantitative amplification of ssDNA (QAOS), ssDNA arising during various cellular processes in complex genomes can be quantified at numerous single-copy and repetitive loci. QAOS is a useful tool to gain insights into the cellular processes that involve ssDNA and the roles of proteins in regulating ssDNA production and responses to ssDNA.