2011 HHMI Summer Scholar Participants
Ian Campbell, Carnegie Mellon University
Mentor: John Woolford
Investigating the Role of Pwp1 in Ribosome Assembly
The ribosome is a fundamental example of a molecular machine and can be used to understand how molecular processes drive cellular function. The ribosomal particle is a complex of 80 ribosomal proteins and 4 ribosomal RNAs (rRNA), responsible for translating messenger RNA into protein. Eukaryotic ribosomal structure and function are well understood, yet how these machines are assembled remains unanswered. Assembly of ribosomes begins in the nucleolus where rRNA is transcribed and continues with addition of ribosomal proteins and processing of rRNA, as the particle travels through the nucleoplasm and into the cytoplasm. Proteins termed “assembly factors” transiently associate with preribosome during this time to assist in assembly. Saccharomyces cerevisiae is used as a model system to study eukaryotic ribosome assembly. I have used this model to elucidate the function of Pwp1; a conserved, non-essential, nucleolar protein that interacts physically and genetically with previously characterized assembly factors. To determine when Pwp1 associates with preribosomes we have assessed which rRNAs, ribosomal proteins and assembly factors copurify with Pwp1. To clarify what role Pwp1 performs in assembly we have knocked-out Pwp1. We will determine the effect of depletion of Pwp1 on assembly factors and ribosomal proteins by affinity purifications coupled with western blots. The depletion strain will be visualized by fluorescent microscopy to determine how intracellular transit of assembling ribosomes is affected by removal of Pwp1. The function of Pwp1 may also be better understood through its structure. Pwp1 contains WD40 repeat domains, consistent with protein-protein and protein-RNA interactions. We will examine how Pwp1’s structure relates to its function by site directed mutagenesis within and surrounding the repeat domains. Determining the function of Pwp1 will add to our knowledge of ribosome assembly. Understanding ribosome biogenesis will help treat ribosome related genetic diseases, target antibacterial drugs, and shape our understanding of molecular machines.
Lianne Cohen, Carnegie Mellon University
Mentor: Jonathan Jarvik
Directed Evolution of Fluorogen-Activating Proteins for Cytoplasmic Expression using Somatic Hypermutation in Ramos Cells
Fluorogen-activating proteins (FAPs) are a new technology used to study protein localization in live cells. Small molecules called fluorogens bind to the FAP, producing a strong fluorescence signal. However, due to their immunoglobulin structure most FAPs fail to fold properly when targeted to the mammalian cytoplasm, which leaves the fluorogen deactivated. The purpose of this project is to directly evolve FAPs in Ramos cells, a human B-cell line, to produce variants capable of functioning in the cytoplasmic environment of mammalian cells. Ramos cells undergo somatic hypermutation, mutating their genes at rates of up to 1/103 nucleotides per generation. Fusion constructs were made with three different FAPs, dNC138, HL4-MG and HL1.0.1-TO1, which are expressed on the cytoplasmic side of the plasma membrane and connected via a transmembrane domain to an extracellular epitope tag. HL4-MG was selected for higher signal using the fluorogen, malachite green, while dNC138 and HL1.0.1-TO1 were mutated to bind a cyano-thiazole orange fluorogen. Iterative steps of mutation by the Ramos cells and fluorescence-activated cell sorting (FACS) were used to generate diversity and select for functional FAPs. The external epitope tag and a fluorophore-conjugated primary antibody were used to monitor the fusion construct expression. Genomic DNA from populations containing mutated FAPs has been isolated and the gene of interest was amplified by PCR. These genes will be sequenced for analysis. We hope to develop a variety of FAPs that produce strong intracellular fluorescence to tag proteins in the cytoplasm of live mammalian cells.
Simone Costa, Carnegie Mellon University
Mentor: Ora Weisz
Role of Endolyn in Pronephric Zebrafish Kidney Development
Endolyn is a transmembrane protein of the sialomucin family, and is expressed in both developing and in adult mammalian tissues. Previous studies have implicated endolyn as having a role in kidney development. Using the model organism Danio rerio we will study the role of endolyn in zebrafish kidney development by knocking down and rescuing the protein. Complete knockdown of endolyn expression in zebrafish embryos using morpholinos leads to visible body deformities including hydrocephaly and body axis curvature as well as defects in renal function, as demonstrated by delayed renal clearance of injected dye and pericardial edema. The wild type phenotype is fully restored by heterologous expression of rat endolyn. Mutations have been made in the different domains of rat endolyn to determine which domain is sufficient and required for efficient phenotype rescue. One particular region of interest is the FIGGI sequence within the transmembrane domain, as it is completely conserved among species and in other related sialomucin proteins. I will be designing and generating mutations within the FIGGI region of rat endolyn and determining whether these mutations rescue the endolyn knockdown phenotype in zebrafish. I will accomplish this by comparative microscopy of the embryos treated with and without the rescue endolyn RNA. I will also stably express the rescue constructs in polarized Madin-Darby canine kidney cells (MDCKs) to determine how the mutations affect the trafficking route by using domain-selective biochemicalassays and indirect immunofluoresence.
Chelsea Weber, Carnegie Mellon University
Mentor: Aaron Mitchell
Functional Analysis of Essential Genes in Candida albicans
This project aims to study essential protein kinase genes in the diploid pathogen Candida albicans by using the Decreased Abundance by mRNA Perturbation (DAmP) allele method. Protein kinases were chosen as gene targets as they frequently are valuable therapeutic targets. This method creates alleles that have the 3’ noncoding region of the gene of interest switched from the wild type sequence to a bacterial sequence. This bacterial sequence lacks information for poly adenylation, transcription termination, and other signals that give stability to the mRNA. Without this stability, there is a decrease in the amount of mRNA and amount of protein produced. This decreased amount is expected to be enough to maintain viability, but also different enough from the wild type to have a noticeable phenotype. My project has focused on protein kinases that we postulated to have a role in cell wall regulation, as the cell wall is an important factor in therapeutic treatments for C. albicans infections. The first five genes to be altered to be DAmP homozygous were SNF1, IRE1, YPK1, DBF2, and ORF19.5376. These alleles were generated from homologous recombination with the DAmP bacterial sequence and URA3 marker. UAU1 cassettes are then inserted into the URA3 gene, and homozygous strains are generated from homologous recombination events with the ura3 sequences and selected for on –ARG –URA media. Out of the initial five genes, homozygotes of SNF1, IRE1, DBF2 and ORF19.5376 were generated. This is approximately consistent with the success rate established for S. cerevisiae (~87%). Reduced levels of mRNA have been confirmed by RT-PCR for SNF1 and DBF2. Surprisingly, increased levels of IRE1 and ORF19.5376 mRNA were found. Phenotypic assays of these four strains are ongoing to further characterize the functions of these genes. Lastly, the library of DAmP strains will be expanded.