Grant Number :    1F32GM064867-01

Pricipal Investigator : Jaglo Kirsten

Project Title : Isolation of Human Defensin Genes

Abstract : Humans are remarkably good at fighting off attacks by pathogenic microorganisms unless they are immunosuppressed. Defensins are innate nonspecific, antimicrobial peptides that disable microbes and some viruses within 30 minutes. To date, six alpha- and three beta-defensins have been isolated in humans. Isolation of additional defensin genes and examination of their antimicrobial activity would increase understanding of the immune system and could be used in cancer treatment. Therefore, I will (1) develop screening conditions to isolate new defensins using the bakers yeast, Saccharomyces cerevisiae; (2) identify and characterize new defensin genes; and (3) further characterize known defensin genes. I will use a novel screening approach where peptides will be secreted from a high secretion strain of S. cerevisiae onto lawns of Gram-negative and Gram-positive bacteria. cDNAs from human tracheal cells will be cloned into a high copy vector containing both the promoter and secretion sequences from the yeast alpha-factor gene, MF-alpha-1. Yeast colonies that are surrounded by reduced bacterial growth will be isolated and the cDNA sequenced. Novel peptides will be examined for specific antibacterial and antifungal activity and tissue-specific expression in humans or mice. To better understand the mechanism of defensin action, known antimicrobial peptides will be randomly mutagenized by PCR, expressed and secreted by yeast (as above), then used to challenge Gram-negative and Gram-positive bacteria. Using optimal and sub-optimal conditions (e.g., high salt concentrations) peptides with increased antimicrobial activity will be isolated and sequenced. Through these experiments I hope to both isolate new defensins and increase understanding of known defensins.


Duration of Award : 01 APR 2002

Amount :

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