Isolation of yeast genes that suppress the chromosome loss defect of 'YAC stability in mitosis' mutants.

The eukaryotic cell cycle and processes that maintain genome stability occur with high fidelity. Mutations in genes important for the structure, replication, repair, and/or segregation of chromosomes are correlated with genome instability (i.e., chromosome rearrangements, increased mutation rates, aneuploidy, abnormal chromatin structure, and/or abnormal gene expression). As part of an effort to identify proteins important for genome stability, we previously implemented a genetic screen in the yeast Saccharomyces cervisiae that allows for visual detection of mutants with increased loss of an ADE2-marked yeast artificial chromosome (YAC). This screen resulted in 132 –AC stability in mitosis (ysm) mutants. Three mutants, ysm76, ysm83, and ysm84, have been further characterized for phenotypes related to genome instability. In addition, both high copy and single copy suppressors of these mutants’ YAC loss defects are being isolated. Identification of these suppressors will contribute to our understanding of protein networks and processes important for eukaryotic genome stability

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

Complementary approaches to identify genes important for chromosome segregation

Faithful transmission of chromosomes during cell division is essential to the functioning of the eukaryotic cell. In humans, errors during chromosome segregation are correlated with diseases such as Down Syndrome and cancer. To elucidate the genes and processes required for accurate chromosome segregation, two types of genetic approaches using the model eukaryotic organism Saccharomyces cervisiae (baker’s yeast) are being implemented. Both approaches rely on an assay system in which the segregation of a non-essential yeast artificial chromosome (YAC) is monitored. The first approach begins with the isolation of mutants with defective chromosome segregation and seeks to identify the gene(s) responsible for producing the mutant phenotype. Four previously isolated YAC stability in mitosis mutants (ysm’s 22, 77, 83 and 84) were characterized as having a marked increase in YAC loss. To identify genes that suppress the YAC loss phenotype in these mutant strains, the ysm mutants were transformed with a yeast genomic plasmid library and screened for plasmids that suppress the mutant phenotype. The suppressor candidates were re-screened, and the specific genes responsible for the suppression are currently being determined. The second approach begins with a known mutation on chromosome segregation. Five genes of known function that are suspected to be important for accurate transmission of chromosome (MRC1, MRE11, MUS81, RAD9, and RAD27) are targeted in this analysis. Following deletion of these genes in a yeast strain containing a YAC, the loss rate of the YAC will be experimentally determined in the deletion mutants and compared to the loss rate in a wild-type strain. These investigations are expected to reveal genes important for the process of chromosome segregation

A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1∶106 with E. coli O157:H7 and affinity constants ranged from 1.57×1010 to 2.79×1010 L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food

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

"Genetic analysis of the human disease-causing gene ATM in yeast Tel1 and Mec1 mutants"

Ataxia-telangiectasia (A-T) is a recessive human disorder characterized by hypersensitivity to radiation, elevated risk of cancer development, nervous degeneration, premature senescence, and immune deficiencies. A-T is caused by mutation of the ATM gene located on human chromosome 11. The large protein product of ATM is a member of the phosphatidylinositol-3-kinase (PI3K) protein family. PI3K's are important for cellular response to DNA damage and are involved in DNA repair, genetic recombination, apoptosis, and cell cycle regulation. Human protein ATR and yeast proteins Mec1 and Tel1 are also PI3K's. Yeast tel1 mutants display chromosome instability and telomere shortening, and yeast mec1 mutants display sensitivity to DNA damaging agents, checkpoint misregulation, chromosome loss, and chromosome rearrangement. The defects observed in human cancer cells are similar to those observed in yeast mec1 and tel1 mutants. Previous research has shown that the human ATR protein can functionally complement the radiation sensitivity of a yeast mec1 mutant. Similarly, yeast tel1 p can partially functionally complement the human A-T phenotype, reducing recombination, apoptosis, and telomere shortening in A-T cells. However, Tel1 p does not reverse the A-T defects in cell cycle checkpoints and sensitivity to radiation, suggesting that these two processes may be regulated by Mec1 p. To determine whether the human ATM gene can functionally complement mec1 and tel1 in yeast, the full length ATM gene and ATM PI3K domain are being cloned and tested for functional complementation of tel1 and mec1 defects.Drake University, College of Arts and Sciences, Department of Biology

Participation of Students in Cooperative Education Programs: A Comparison of Students With and Without Disabilities

132 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.This study described the types of jobs students with and without disabilities acquired through cooperative vocational education programs while in high school. Findings indicated significant correlations between the jobs attained through the cooperative vocational education program and prospective wages, hours, and total job openings available to students in the general labor market subsequent to the training they received through their cooperative program. Further, the study found significant relationships between student characteristics (e.g., gender, ethnicity and geographical location) and the types of cooperative vocational educational jobs they were participating in, as well as the prospective hourly wages, weekly hours, and total job openings that students could expect to attain in the general labor market based on the training they received from their cooperative vocational program.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Analysis of mRNA changes as a function of learning and methamphetamine exposure

Methamphetamine is a powerful, addictive drug that is of great concern in Iowa and the Midwest. Powerful methamphetamine abuse is correlated with depression and learning disabilities (e.g. attention deficit disorder). Although the mechanism of action of methamphetamine in the brain has been well characterized at the cellular and synaptic level, the effects of this drug at the genetic level are not well understood. To analyze the possible effects of methamphetamine on learning, we are using real-time PCR to investigate changes in gene expression (i.e., messenger RNA levels) in rat brains as a function of learning and exposure to methamphetamine. Specifically, we are analyzing relative mRNA levels from genes whose products have been implicated in learning and/or addiction: GABA receptors, dopamine receptors, and glutamate (NMDA) receptors. Comparison of these data sets is expected to reveal potential cellular and molecular mechanisms involved in producing drug-induced learning disabilities