1999 Summer HHMI-supported Participants-HHMI Undergraduate Program - Carnegie Mellon University

1999 Summer HHMI-supported Participants

Rebecca Frederick

Rebecca Frederick, Carnegie Mellon University
(Advisor: Dr. Elizabeth Jones)

Cloning of Green Fluorescent Protein downstream of the promoter of CAR1 in yeast

In the bakers yeast Saccharomyces cerevisiae, mitogen activated protein (MAP) kinase pathways function to transmit signals from receptors at the plasma membrane and activate transcription via a series of signal transducers. There are six MAP kinases in yeast, all of which respond uniquely to particular stimuli, but the pathways which activate each as well as the transcription factors downstream of these MAP kinases, share many components. Thus, yeast is a model organism for investigating the cross-talk and regulation between different MAP kinase pathways. There is one MAP kinase pathway that responds to nitrogen deficiency, causing a change in growth patterns. When starved for nitrogen, the cells release vacuolar stores of arginine and activate transcription of CAR1, the gene for arginase. Arginase hydrolyzes arginine so that its nitrogens may be used during amino acid synthesis. It has been shown that an upstream activation sequence of CAR1 (pCAR1) is required for CAR1 transcription. To investigate the relationship between CAR1 transcription and the MAP kinase pathways, I have cloned superglow green fluorescent protein (sGFP) in frame downstream of pCAR1 using standard molecular cloning techniques. This construct has been integrated into the genome of yeast at the pCAR1 site such that there are now two functional copies of pCAR1, one followed by CAR1, one by sGFP. Thus when subjected to nitrogen starvation or arginine rich medium, the cells should make both sGFP and arginase. This reporter gene construct will be used to evaluate a genome-wide mutagenesis screen for genes involved in the CAR1 pathway, as well as showing the effect of disrupting genes involved in the MAP kinase pathways.

Michelle Griffin

Michelle Griffith, Carnegie Mellon University
(Advisor: Dr. Amy Csink)

Establishment and Characterization of a New Transgene Insertion near the brown locus of Drosophila melanogaster

Heterochromatin has long been known to be involved in position-effect variegation (PEV), the silencing of a gene normally found in euchromatin due to its relocation to an area within or proximal to heterochromatin. PEV has been studied extensively, and in recent years it has become apparent that heterochromatin has more varied and widespread effects than previously thought. The Csink lab is investigating a particular PEV phenomenon, trans-inactivation, caused by the spatial association of heterochromatic compartments in Drosophila melanogaster. The mechanism and properties of trans-inactivation can be studied by introducing transgenes into or near loci that have been shown to be subject to trans-inactivation, such as the brown (bw) eye color locus.

Lindsay Jorgensen

Lindsay Jorgensen, Carnegie Mellon University
(Advisor: Dr. Robert F. Murphy)

The Role of Na+,K+-ATPase Isoforms in Endosomal pH Regulation

Endosomal pH plays a crucial role in the dissociation of receptor-ligand complexes. Na+,K+-ATPase has previously been found to have a role in endosomal pH regulation. Cells with an endosomal pH of 6.0-6.2, Class H cells, have been found to express only one isoform of the beta subunit of their Na+,K+-ATPase, beta 1. Cells with an endosomal pH of 5.4, Class L cells, express beta 1 and beta 2 isoforms. My goal was therefore to test whether expression of the beta 2 isoform is sufficient for exhibiting a low endosomal pH. PCR primers with engineered restriction enzyme sites were designed in order to amplify the entire coding region of the beta 2 cDNA. These primers were used to amplify beta 2 from a human brain cDNA library and the nature of the fragment was confirmed by sequencing. It was then inserted into a pCMV-Tag3 vector which contained a N-terminal c-myc epitope tag. In future wark the plasmid will be transfected into A549 cells, a class H cell line. The presence of the beta 2 isoform will be verified using an antibody to c-myc. Once the presence of the isoform is confirmed the endosomal pH will be analyzed using fluorescently-labelled transferrin. The pH analysis will determine whether the beta 2 isoform can create a class L phenotype in class H cells. This will clarify the role of the Na+,K+-ATPase in endosomal pH regulation.

Kristina Lamothe

Kristina Lamothe, Carnegie Mellon University
(Advisor: Dr. Elizabeth Jones)

Mutagenesis of VPS16 Gene required for Vacuole Biogenesis in Yeast

In Sacchromyces cerevisiae (baker's yeast), proteins produced in the ER are transported within the cell via vesicle mediated transport pathways. These pathways are aided by several proteins. Vps16p is known to form an oligomeric complex with Vps33p, Pep3p, and Pep5p. This complex is known to aid in a late step in transport, but the exact function of Vps16p is unknown. The goal of this project was to create temperature sensitive Vps16 mutants so that VPS16 can be studied. To do this, a null mutant was first created. The null mutant was created by making a construct in which the VPS16 gene had been disrupted with a gene for tryptophan production. The disrupted gene was then removed from the construct and transformed into competent yeast cells. Cells in which this DNA fragment transplaced the wild type copy VPS16 in the genome were then selected for. This null mutant is now ready to uptake mutated forms of VPS16. In the fall when I continue my research, I will incorporate mutated forms of the gene which I have already made by transforming the normal gene into a mutagenizing strain of E. coli. The mutants can then be characterized to see which pathways are affected. This can be done by observing the presence or lack thereof of certain proteins which are known to be used within the main vacuole of the yeast and are known to be modified during transport to the vacuole. When a protein is not present in its modified form, it can be concluded that the pathway which is supposed to transport that protein is not functioning. Hopefully, the results of this study will add to what is known about transport within a yeast cell. The information gathered on yeast can be generalized and applied to many other life forms.

Joshua Mugford

Joshua Mugford, Carnegie Mellon University
(Advisor: Dr. Charles Ettensohn

GSK-3b 5' Endpoint Splicing Variants in Lytechinus variegatus

It has been seen that there are many similarities among the development of many different species, involving both the structure of the embryo and the proteins involved in the regulation of development. GSK-3b is such a protein. Researching these conserved proteins in other species could help lead to a better understanding of embryological development of the animal kingdom, including humans. Understanding the development of humans could help in numerous areas, one being medical research. This project will involve the studying of the five prime DNA endpoints of the gene encoding GSK-3b using DNA of the sea urchin genus Lytechinus variegatus. The primary goal is to detect if this genus has variation at the five prime endpoint, as was seen in separate study of a different genus of sea urchin (Emily-Fenouil et al., 1998). PCR techniques will be used to isolate and amplify the five prime untranslated regions of GSK-3b from a cDNA library of L. variegatus. If these differences are seen, functional correlation will be investigated by using Northern and Southern blot analysis and whole mount in situ analysis. The combination of these three tests will give information about during what stage of development, where in the embryo the protein is expressed and how many copies of the gene are present in the genus of the sea urchin. It is expected that three different variations will be found. In addition to the investigation of the enpoint variations, a antibody to GSK-3b will also be developed.

Miguel Nogueras

Miguel Nogueras, University of Puerto Rico, Mayaguez, PR
(Advisor: Dr. Susan Henry)

The in vivo Effect of a mutation at a phosphorylation site in the Opi1 gene regulator

In the yeast Saccharomyces cerevisiae, the biosynthesis of phospholipids is regulated in response to the levels of precursors such as inositol and choline. INO1 is the most highly regulated of the structural genes that are repressed in response to inositol and choline in the cell. Opi1p negatively regulates the INO1 gene and deletion of OPI1 gene causes the overproduction of inositol. Three signal transduction pathways in yeast have been shown to affect expression of INO1. The protein kinase C (PKC) pathway is one of these. It has been shown in-vitro, that an Opi1p peptide containing Ser26 is phosphorylated by rat brain Pkc1p. A site-directed opi1 mutant gene (OPI1-1) with a change from Ser26 to Ala was inserted into a CEN based plasmid, pRS313. Wild type (W303) and opi1D mutant (JAG1) yeast strains were transformed with pRS313, pRS313OPI1 and the mutated pRS313OPI1-1. These tranformants were assied for their Opi- phenotype by an Opi test. Preliminary results have shown that the Ser26 to Ala mutation partially reduces the Opi1p activity in-vivo. These results suggest that there is regulation of Opi1p activity by phosphorylation. More studies are necessary to confirm the in-vivo phosphorylation of Opi1p.

Andrew Walsh

Andrew Walsh, Carnegie Mellon University
(Advisor: Dr. Adam Linstedt)

Locating the Binding Domains of Various Golgi-associated proteins in Giantin

Giantin is a transmembrane protein associated with the Golgi apparatus that has been shown to bind other Golgi proteins in vitro, including p115, GM130 and COP-I. All of these proteins are believed to be involved in transport between the endoplasmic reticulum and the Golgi, as well as in intra-Golgi transport. The goal of this project is to determine where each of these proteins binds to giantin. Two techniques, site-directed mutagenesis and proton NMR, will be used to examine the N-terminal portion of giantin. GST-giantin fusion proteins will be synthesized with specific mutations and then assayed for binding with the proteins of interest. Additionally, the structure of the N-terminus will be determined by N15 NMR. It is hoped that determining where these proteins bind will provide information about the role of all of these proteins. For instance, if it is found that two or more of them share a binding site, that would suggest some sort of regulation or competition.

Emily Zajano

Emily Zajano, Carnegie Mellon University
(Advisor: Dr. David Hackney)

Kinetics and Properties of BimC Monomers and Dimers of Varying Lengths

Kinesin is a protein family which is involved in the movement of chromosomes and other organelles along microtubules. Different proteins within the superfamily have similar head motor domains which can be located either at the N-terminal, the C-terminal or integrated into the middle of the protein. BimC is a specific protein that has an N-terminal head domain homologous to conventional kinesin. The monomer is a singular protein unit that when bound to another of the same unit, becomes a dimer. The protein hydrolyzes 1 ATP with each binding and unbinding to the microtubule. The ATPase rate is determined by the rate at which the protein slides toward the plus end of the microtubule. Multiple plasmids were constructed with varying lengths of the BimC motor domain. The observed ATPase rates on the shortest monomer and dimer proteins indicate that BimC processes ATP almost as rapidly as conventional kinesin. This is unexpected because BimC was not thought to be a processive protein. Kinesin is processive, using the two head domains to walk hand-over-hand down the microtubule. The BimC protein being used for these experiments does not include the first 72 amino acids of the N-terminal region of the protein. When the ATPase assay was performed on the BimC monomer including the first 2 amino acids, the rate of hydrolysis slowed down. This suggests that the first 72 amino acids may bind the protein more tightly to the microtubule. Thus ATP is not hydrolyzed as easily and the rate slows down. Further research would include the previous experiments performed on the dimer version of the protein with the first 72 amino acids and the full length BimC both with and without the first 72 amino acids. MantATP fluorescence assay could also be conducted for more accurate results.