Identification of putative interactions between swine and human influenza A virus nucleoprotein and human host proteins

Abstract Background Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies. Methods In this study, the NP from a swine IAV was cloned into a yeast two-hybrid “bait” vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This “bait” was used to screen a Y2H human HeLa cell “prey” library which consisted of human proteins fused to the Gal4 protein’s activation domain (AD). The interaction of “bait” and “prey” proteins resulted in activation of reporter genes. Results Seventeen positive bait-prey interactions were isolated in yeast. All of the “prey” isolated also interact in yeast with a NP “bait” cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B). Conclusions Ten human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.http://deepblue.lib.umich.edu/bitstream/2027.42/110223/1/12985_2014_Article_228.pd

Isolation and Characterization of Saccharomyces cerevisiae Mutants Defective in Chromosome Transmission in an Undergraduate Genetics Research Course

An upper-level genetics research course was developed to expose undergraduates to investigative science. Students are immersed in a research project with the ultimate goal of identifying proteins important for chromosome transmission in mitosis. After mutagenizing yeast Saccharomyces cerevisiae cells, students implement a genetic screen that allows for visual detection of mutants with an increased loss of an ADE2-marked yeast artificial chromosome (YAC). Students then genetically characterize the mutants and begin efforts to identify the defective genes in these mutants. While engaged in this research project, students practice a variety of technical skills in both classical and molecular genetics. Furthermore, students learn to collaborate and gain experience in sharing scientific findings with others in the form of written papers, poster presentations, and oral presentations. Previous students indicated that, relative to a traditional laboratory course, this research course improved their understanding of scientific concepts and technical skills and helped them make connections between concepts. Moreover, this course allowed students to experience scientific inquiry and was influential for students as they considered future endeavors

"Identification of yeast genes important for chromosome transmission"

Accurate transmission of chromosomes during cell division requires that chromosomes are fully replicated and free of damage prior to cell division. Furthermore, cellular processes that separate chromosomes must be functional. As part of an effort to identify proteins important for chromosome transmission, we implemented a genetic screen in the yeast Saccharomyces ceresisiae that allows for visual detection of mutants with an increase in the loss of an ADE2-marked yeast artificial chromosome (YAC). This screen resulted in 132 YAC stability in mitosis (ysm) mutants. In addition to genetic characterization of these mutants, subsets of the ysm's were analyzed for YAC loss rate, sporulation efficiency, forward mutation frequency, and cell morphology. Mutants were also tested for sensitivity to high salt, caffeine, hydroxyurea, and benomyl. The results elucidated mutant phenotypes that will facilitate cloning the wild-type genes that are altered in these mutants. To this end, high copy suppressors of ysm83 and ysm84 have recently been isolated.Drake University, College of Arts and Sciences, Department of Biology