2000 Summer Scholar Participants-HHMI Undergraduate Program - Carnegie Mellon University

2000 Summer HHMI-supported Participants

image of Alexander Bounoutas

Alexander Bounoutas, Carnegie Mellon University
(Advisor: Dr. Amy Csink)

Analysis of trans-inactivation using lines generated by P-element replacement

BrownDominant (bwD) is an allele of the brown eye-color gene in Drosophila melanogaster that contains an insertion of heterochromatin at the distal tip of the second chromosome. bwD/bw+ flies express bw in only a few ommatidia. The silencing of bw+ by bwD is referred to as trans-inactivation, and is thought to result from the mislocalization of the bw allele to the heterochromatic nuclear compartment following somatic pairing with the bwD allele. In order to study the susceptibility of various promoters to trans-inactivation, we have generated lines that contain different P-element transgenes in 59E, the site of the bw locus. One P-element used, (P{hsp26-pt-T}), contains two reporter genes, white (w) and a transcript from barley, driven by the hsp70 and hsp26 promoters, respectively. The hsp70 promoter drives the w gene even without heat shock. The P-element was inserted into chrw and Dcp-1, two loci in 59E with varying distance from bw. The transgenic lines produced were then crossed with bwD flies to assess the effects on the hsp70 and hsp26 promoters at both loci. The susceptibility of the hsp70 promoter was assessed phenotypically by comparing eye color, and quantification of the level of trans-inactivation of both promoters was determined via northern blotting. It was concluded that both promoters were susceptible to trans-inactivation at the chrw locus, which is located 4.3 kb proximally from the bw gene. Neither promoter was susceptible to trans-inactivation at the Dcp-1 locus, located 23.5 kb distally from bw.

Rachel Buchwalter

Rachel Buchwalter, Carnegie Mellon University
(Mentor: Dr. David Hackney)

Spindle formation and tight binding of microtubules by kinesin tail region

Kinesin tail region constructs bind tightly to microtubules and have been observed to create spindle-like clumps of microtubules. One proposed mechanism for this effect shows kinesin constructs sliding down the microtubule and (unlike the head region), remaining attached once they reach the terminus. Some of the tails then could attach to another microtubule, again sliding along towards the positive end, eventually creating clumps of several microtubules. To determine if this is how the phenomenon occurs a transmission electron microscope was used to study aggregations of kinesin 937 at the ends of single microtubules, before the tail constructs had attached to other microtubules and clumped. As a control, unfolded kinesin constructs (kinesin 894) were added, which should not have bound and clumped microtubules. Results showed kinesin 937 indeed formed aggregates, but 894 also seemed to form smaller aggregates as well.

Michael Gehring

Michael Gehring, Carnegie Mellon University
(Advisor: Dr. Charles Ettensohn)

The Effects of Modulating the Notch Pathway and the b-catenin Pathway on Mesoderm Development in the Sea Urchin

In the sea urchin embryo, all non-skeletogenic mesoderm normally develops from the vegetal part of the embryo. Mesoderm is induced by micromeres, which lie at the vegetal pole of the embryo. Recent findings suggest that mesoderm development may be controlled by two separate molecular pathways, the Notch pathway, and the b-catenin pathway. Activation of Notch, a cell-surface receptor, has been shown to be essential in the formation of mesoderm. Recently, a ligand to this receptor, Delta, has been shown to be expressed by the micromeres. The other pathway involves modulation of gene expression by b-catenin, which induces endoderm and possibly mesoderm, two vegetal cell types. Lithium treatment, which activates the b-catenin pathway, causes "vegetalization" in embryos, resulting in an increase in the amount of endoderm produced, and it has been suggested that the same is true of mesoderm, but there is no quantitative data to support this view.

The purpose of these experiments was to affect the development of non-skeletogenic mesodermal cell types by means of each of these pathways. The goal of the first experiment was to determine if lower levels of Delta lead to a decrease in mesodermal cell development. To accomplish this, variable numbers of micromeres were removed. The resulting embryos were grown through the pluteus stage of development and then were scored for various mesodermal cell types using light and indirect immunofluorescence microscopy. The data obtained showed a clear positive correlation of pigment cells to micromeres, though blastocoelar and muscle cells did not appear to be affected. The goal of the second experiment was to examine whether increases in b-catenin activity cause an increase in mesodermal cells. To test this, embryos were treated with lithium to activate the b-catenin pathway. The resulting embryos were allowed to develop to the pluteus stage, and then were scored for various mesodermal cell types as described above. The data obtained did not show an increase in mesoderm, as expected; however, in the case of pigment cells, a decrease was observed. It is unclear how blastocoelar and muscle cells were affected by treatment with lithium.

Laura Gonzalez

Laura Gonzalez, Carnegie Mellon University
(Advisor: Dr. Peter Berget)

A molecularly accurate animation of RNA transcription initiation

In order to convey an accurate picture of transcription, the latest electron microscopy pictures of RNA Polymerase obtained by Darst's group at Rockefeller University were interpreted into a semi-sophisticated 3D computer animation program (3D Studio MAX 2.5) that could also handle the importation of a large DNA molecule made up of many many spheres. Using the bending and wrapping model of transcription initiation described in Coulombe and Burton, the animation should serve to illustrate one of the latest and most accurate models of transcription initiation. Attention was paid to both scale and placement of the molecules, and which regions actually interact.

Jodi Gureasko

Jodi Gureasko, Carnegie Mellon University
(Advisor: Dr. Gordon Rule)

Co-crystallization of the RNA-binding domain of the Rho Transcription Termination Factor with Oligonucleotides

Rho is an essential protein in E. coli that terminates the transcription of many genes. Rho is a hexameric protein that releases newly synthesized RNA from transcription complexes by a mechanism that requires ATP hydrolysis. Rho binds to single-stranded DNA or RNA with equal affinity, but rho is only activitated when it is bound to single-stranded RNA.

The Rho protein consists of two domains - the RNA-binding domain (residues 1-130), and the ATPase domain (residues 131- 419). To better understand the mechanism of transcriptional termination at the level of protein-nucleic interactions, I have isolated and purified the RNA binding domain of Rho (Rho130). The intent is to grow co-crystals of Rho130 in complex with different DNA and RNA oligonucleotides. Analysis of the co-crystals by x-ray crystallography will provide insight into how Rho interacts with nucleic acids.

Mindy Hebert

Mindy Hebert, Carnegie Mellon University
(Advisor: Dr. Adam Linstedt)

Isolation of a Mutant Chinese Hamster Ovary Cell Line that Mislocalizes a Golgi Membrane Protein to the Cell Surface

We have attempted to isolate a mutant Chinese Hamster Ovary (CHO) cell line that mislocalizes a Golgi membrane protein, GPP130, to the cell surface. It has been shown that a version of GPP130 missing all targeting sequences mislocalizes to the cell membrane. This fact has led us to believe that a genetic mutation affecting the targeting function of the secretory pathway can be isolated by selecting for mutated cells that express Golgi membrane proteins, GPP130 in particular, on the cell surface. A selection procedure known as antibody panning will be used to select for the desired mutants. This panning procedure has proven effective for enhancing a cell population for those cells that express GPP130 on their surface by 25-75%. Mutant cells are also screened with total and surface immunoflourescence against GPP130. CHO cells stably transfected with human GPP130 were mutagenized with ethyl methane sulfonate (EMS). Three trials of mutagenesis were conducted. The second and third trials did not yield any viable cells after one round of selection with antibody panning. The first trial however, has yielded a single mutant clone after two separate rounds of panning. The morphology of this clone is not yet known. If, by immunoflourescence, we determine that the clone expresses GPP130 on its surface, we will begin experiments to characterize it. If the clone is not positive for surface GPP130, we will continue conducting mutagenesis and selection experiments.

Christopher Hodge

Christopher Hodge, Carnegie Mellon University
(Advisor: Dr. Elizabeth Jones)

The Creation of a Ubr1 Delete Strain in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, protein degradation by the ubiquitin pathway controls the intracellular levels of certain regulatory proteins. It has been shown that the gene product of UBR1, the E3 enzyme of a ubiquitin-dependent N-rule pathway, complexes with regulatory proteins in order to activate their degradation via the proteolytic pathway. Specifically, the UBR1 gene product targets proteins at the amino terminals, thereby marking them for degradation. In previous work, UBR1 was isolated from the yeast genome using standard molecular cloning techniques, and subsequently ligated into a plasmid. The insert of UBR1 was modified by an approximate 5 kb deletion of a fragment extending from the 5' end to the 3' end, leaving about 500 bases of homology on either side. Current research has involved inserting selectable markers into the plasmid between the 3' and 5' ends of the ubr1 disruption, and within the deleted portion of the gene. The selectable markers provided for positive conformation of the transformed plasmid. After the ubr1 deletion plasmid was obtained, it was linearized. The linear fragment was then transplaced into yeast where crossing over occurred through a process of homologous recombination with the functional UBR1 gene in the genome, thus creating a ubr1 deletion in the genome. The ubr1 deletion strain will facilitate experimentation concerning the possibility that other proteins, which may complex with the UBR1 gene product, may be involved with protein degradation through a ubiquitin-dependent N-end rule pathway. For instance, the protease B precursor is known to be unstable in pbn1-1 strains due to the presence of an N-terminal amino acid that suggests a substrate that is degraded via the N-rule pathway. Thus, future experiments will involve testing if the protease B precursor is stable in pbn1-1 Δ ubr1 double mutants. If the precursor proves to be stable, it will identify the protease B precursor as the first bonafide substrate of the ubiquitin-dependent N-rule pathway.

Joshua Mugford

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

Characterization of a Discoidin Domain Receptor (DDR), in the Sea Urchin Lytechinus variegatus

The development of an organism is a complex process involving many different pathways. One pathway critical to the development is skeletogenesis (the creation of the skeleton), a process performed by primary mesenchyme cells (PMCs) in the sea urchin. Discoidin Domain Receptor (DDR) is a highly conserved trans-membrane tyrosine kinase receptor thought to function in skeletogenesis using collagen as a signal. The extracellular collagen is bound either directly or indirectly by the discoidin domain, stimulating the activity of the intracellular tyrosine kinase domain. A sequence of cellular events essential to the formation of the skeleton is thought to commence due to these phosphorylation episodes. Previous studies have shown collagen as an essential molecule in skeletogenesis. If similar phenotypes are seen in embryos that have lost DDR function, a link could be established among DDR, collagen and skeletogenesis. A clone of DDR from Lytechinus variegatus has been isolated as a result of a large scale cDNA sequencing project.

The goal of this project is to characterize DDR in the sea urchin Lytechinus variegatus using various techniques. Polyclonal antibodies against non-overlapping fragments of the protein can be developed and used in western blotting and immunostaining to help determine when and where the protein is present in the embryo. Whole mount in situ hybridizations can be used to identify when and where the message of DDR is being transcribed. The function of the protein can also be masked in embryos through the use of anti-sense oligonucleotides and the expression of a dominant negative from of DDR. Observing the phenotypes of these embryos will help determine if DDR is involved in the formation of the skeleton. At this point, three fragments of DDR, each corresponding to a different part of the protein, have been cloned and expressed as protein constructs. One fragment is ready for rabbit polyclonal antibody production, while the other two are in the process of purification. Once these antibodies are raised against DDR, immunostaining can be performed on embryos at different stages of development. Each of the techniques mentioned above, combined with DNA sequence analysis can be used to fully characterize DDR within the sea urchin embryo, eventually leading to the further understanding of skeletogenesis. Due to the high conservation of this protein within nature, further elucidation of the function of DDR in other systems may have implications for the further understanding of this protein within humans.