Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT

Ethanol: Salvation or Damnation?

Contents: Opening Essay BY JOE STARITA Running on Empty: Food vs. Fuel BY MIMI ABEBE The recent boom in the ethanol industry has resulted in a greater demand for corn, which, in turn, has led to a strain on the supply, triggering higher prices Nightmares in the Ethanol Dream BY CASSIE FLEMING Politicians are quick to endorse the benefits of ethanol production. Others say that view is an illusion. Follow the Maize BY MIMI ABEBE The step-by-step stages of ethanol production History of Ethanol BY MIMI ABEBE Ethanol’s controversial history dates back to the 1800s Precise Portions BY CASSIE FLEMING Precision farming assists farmers in meeting government mandates for increased corn-ethanol production Subsidies Support Ethanol BY AARON E. PRICE The ethanol industry receives subsidies from every level of government Unlimited Resource or Looming Roadblock? BY AARON E. PRICE Conflicting needs for water complicate Nebraska’s effort to meet ethanol production goals Increased Corn Production Can Pollute Water BY AARON E. PRICE As farmers raise more corn for ethanol, they’re likely to use more nitrogen fertilizer and other chemicals that can pollute water Corn Monoculture No Friend of Biodiversity BY AARON E. PRICE Federal mandates for corn ethanol, which encourage farmers to plant more corn, may threaten the biodiversity of grasslands Honing in on the Homeland BY CASSIE FLEMING In 2006, the United States was the world’s top energy user Ethanol and the Elements BY MELISSA DROZDA and CAROLYN JOHNSEN Using ethanol in place of gasoline helps to reduce carbon dioxide emissions, but this solution may contribute to the problems New Deal or No Deal BY LUCAS JAMESON Minden’s city officials hope their ethanol plant will spark long-term economic growth Genesis on Hold BY LUCAS JAMESON The Genesis plant filed for bankruptcy, leaving millions of dollars owed to companies and local farmers Infrastructure Impacts BY CAROLYN JOHNSEN How will ethanol be moved around — from factory to distributor to vehicle? Sweet Ideas: Alternative Biofuels BY LUCAS JAMESON Scientists and farmers alike promote research of alternative energy crops but without necessary funds the search to find a cost-effective method continues Reporters, Photographers, Graphics, Documentary, Editors, Designers, Facult