2011 SRI Participants-HHMI Undergraduate Program - Carnegie Mellon University

2011 Summer Research Institute (SRI) Participants

Arshia Ahuja

Arshia Ahuja, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

In vivo Detection of Cyclic AMP Signaling in Candida albicans

Candida albicans (C. albicans) is a form of yeast that is most commonly found in the mucosal linings of the gastrointestinal and genitourinary tracts in the human body and under normal circumstances is harmless. However, it has the ability to cause dangerous opportunistic infections in immunocompromised patients. Specifically, C. albicans can form biofilms on medically implanted devices, such as catheters and artificial joints. These biofilms are comprised of a mesh of oval shaped yeast cells and tubular hyphal cells, both of which are extremely important for the proper formation of the biofilm (Mitchell et al.). Much is known about the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway, which governs the formation of hyphal cells in C. albicans (Park et al.). Interactions among the C. albicans cAMP pathway components Bcy1, Tpk1, Tpk2, and Efg1 have been studied extensively, but a detailed analysis of the physical interactions among these proteins within a biofilm has not been performed. We created fusion proteins designed for in vivo physical interaction assays for the Bcy1-Tpk1/2 and Tpk1/2-Efg1 complexes. The TPK1 and TPK2 genes were tagged with RFP and integrated into the C. albicans genome. The EFG1 and BCY1 genes fused to the SEP7 gene. Plasmids expressing these Sep7 fusions were transformed into the Tpk1-RFP or Tpk2-RFP expression strains. Interactions between these fusion proteins can then be observed in C. albicans cells and biofilms via fluorescence microscopy. These results should give us a better understanding the role of cAMP-PKA pathway in the formation of C. albicans biofilms.

Victor Bass

Victor Bass, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

In vivo Detection of Cyclic AMP Signaling in Candida albicans

Candida albicans (C. albicans) is a form of yeast that is most commonly found in the mucosal linings of the gastrointestinal and genitourinary tracts in the human body and under normal circumstances is harmless. However, it has the ability to cause dangerous opportunistic infections in immunocompromised patients. Specifically, C. albicans can form biofilms on medically implanted devices, such as catheters and artificial joints. These biofilms are comprised of a mesh of oval shaped yeast cells and tubular hyphal cells, both of which are extremely important for the proper formation of the biofilm (Mitchell et al.). Much is known about the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway, which governs the formation of hyphal cells in C. albicans (Park et al.). Interactions among the C. albicans cAMP pathway components Bcy1, Tpk1, Tpk2, and Efg1 have been studied extensively, but a detailed analysis of the physical interactions among these proteins within a biofilm has not been performed. We created fusion proteins designed for in vivo physical interaction assays for the Bcy1-Tpk1/2 and Tpk1/2-Efg1 complexes. The TPK1 and TPK2 genes were tagged with RFP and integrated into the C. albicans genome. The EFG1 and BCY1 genes fused to the SEP7 gene. Plasmids expressing these Sep7 fusions were transformed into the Tpk1-RFP or Tpk2-RFP expression strains. Interactions between these fusion proteins can then be observed in C. albicans cells and biofilms via fluorescence microscopy. These results should give us a better understanding the role of cAMP-PKA pathway in the formation of C. albicans biofilms.

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Molly Berntsen, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Required Domains of APC2 for Cortical Localization in Drosophila Melanogaster

Adenomatous polyposis coli (APC) is a protein found in higher eukaryotes. It functions as a negative regulator of Wnt signaling and it promotes stability of microtubules in the cytoskeleton. Too much Wnt signaling can cause cancer initiation and instability of the cytoskeleton can cause cancer progression. For this reason, APC acts as a tumor suppressor in humans and insufficient and/or dysfunctional APC is linked to over 80% of colon cancer cases. Drosophila melanogaster have two versions of APC, APC1 and APC2. It has been reported that APC2 localizes to the cortex of the cell through means of N-terminal Armadillo repeats and a novel C-terminal domain named C30 (McCartney et al. 2011). To study the specific residues in C30 that are required for cortical localization of APC2, we transfected Drosophila S2 cells with APC2 mutant constructs containing only the Armadillo repeats and subdomains of C30. Here, we show that only the Armadillo repeats and a subdomain of C30 are required for cortical localization of APC2. Within this subdomain of C30 lies a highly conserved region to many species of Drosophila. We hypothesize that this conserved region binds to the cortex through coiled-coil protein interactions. To disrupt coiled-coil formation, we propose a scheme in which hydrophobic residues found in the conserved region are changed to a short side-chain, polar amino acid such as threonine.

Christie Cutting

Christie Cutting, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Mutant EcoRV as a Model System

Studies of the specific binding of the restriction endonuclease EcoRV to DNA can give valuable information on the specific binding of other proteins to DNA, such as polymerases and repressors. Most studies of EcoRV have been done using protein crystallization, but nuclear magnetic resonance (NMR) can give dynamic data because it is a solution phase technique. To this end we worked toward making mutant EcoRV protein that should not bind the metal ions necessary for cleavage of the DNA but should still bind the recognition sequence specifically. Such mutants would be perfect for NMR studies of EcoRV because they should remain bound to their recognition sequence instead of undergoing the extremely quick cleavage reaction and subsequently changing conformation. In the course of our research we were able to produce mutant genes for EcoRV through mutagenic PCR and subsequently insert some of those genes into final pET expression vectors for protein production. Studies of these mutant proteins could potentially provide information not only about the wild-type EcoRV but also about all proteins that bind specifically to DNA. Increased knowledge about such proteins could be useful across the field of biology (to improve PCR efficiency, gene therapy, etc.).

Tomas Dardet

Tomas Dardet, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Ribosome Assembly in Saccharomyces cerevisiae: Investigating genetic interactions among Nop12, EBP2, and BRXI

Ribosomes are cellular components made up of 60s and 40s subunits that come together to act as protein factories inside living cells, catalyzing the reactions that join together individual amino acids to create polypeptides essential for the survival of that cell. The DNA of living cells codes for proteins that act as assembly factors that work in concert to assemble the ribosomes’ constituent parts: ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). These assembly factors have been the focus of intense research in recent years and over 200 assembly factors have been found to participate in the multi-faceted process of ribosome assembly. The goal of our research is to investigate the genetic interactions among three known assembly factors known to be involved in splicing of the 25s rRNA within 60s ribosomal subunit assembly: Nop12, EBP2 and BRXI. To this end, we made mutations wherein a selectable marker was integrated at the Nop12 site in either wild type or mutant BRXI or Ebp2 S. cerevisiae candidate strains. This method produced cold sensitive phenotypes in the Nop12 deleted mutants, and will allow for further genetic analysis of these double mutants.

Paige Davison

Paige Davison, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Required Domains of APC2 for Cortical Localization in Drosophila Melanogaster

Adenomatous polyposis coli (APC) is a protein found in higher eukaryotes. It functions as a negative regulator of Wnt signaling and it promotes stability of microtubules in the cytoskeleton. Too much Wnt signaling can cause cancer initiation and instability of the cytoskeleton can cause cancer progression. For this reason, APC acts as a tumor suppressor in humans and insufficient and/or dysfunctional APC is linked to over 80% of colon cancer cases. Drosophila melanogaster have two versions of APC, APC1 and APC2. It has been reported that APC2 localizes to the cortex of the cell through means of N-terminal Armadillo repeats and a novel C-terminal domain named C30 (McCartney et al. 2011). To study the specific residues in C30 that are required for cortical localization of APC2, we transfected Drosophila S2 cells with APC2 mutant constructs containing only the Armadillo repeats and subdomains of C30. Here, we show that only the Armadillo repeats and a subdomain of C30 are required for cortical localization of APC2. Within this subdomain of C30 lies a highly conserved region to many species of Drosophila. We hypothesize that this conserved region binds to the cortex through coiled-coil protein interactions. To disrupt coiled-coil formation, we propose a scheme in which hydrophobic residues found in the conserved region are changed to a short side-chain, polar amino acid such as threonine.

Sarah Horner

Sarah Horner, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

In vivo Detection of Cyclic AMP Signaling in Candida albicans

Candida albicans (C. albicans) is a form of yeast that is most commonly found in the mucosal linings of the gastrointestinal and genitourinary tracts in the human body and under normal circumstances is harmless. However, it has the ability to cause dangerous opportunistic infections in immunocompromised patients. Specifically, C. albicans can form biofilms on medically implanted devices, such as catheters and artificial joints. These biofilms are comprised of a mesh of oval shaped yeast cells and tubular hyphal cells, both of which are extremely important for the proper formation of the biofilm (Mitchell et al.). Much is known about the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway, which governs the formation of hyphal cells in C. albicans (Park et al.). Interactions among the C. albicans cAMP pathway components Bcy1, Tpk1, Tpk2, and Efg1 have been studied extensively, but a detailed analysis of the physical interactions among these proteins within a biofilm has not been performed. We created fusion proteins designed for in vivo physical interaction assays for the Bcy1-Tpk1/2 and Tpk1/2-Efg1 complexes. The TPK1 and TPK2 genes were tagged with RFP and integrated into the C. albicans genome. The EFG1 and BCY1 genes fused to the SEP7 gene. Plasmids expressing these Sep7 fusions were transformed into the Tpk1-RFP or Tpk2-RFP expression strains. Interactions between these fusion proteins can then be observed in C. albicans cells and biofilms via fluorescence microscopy. These results should give us a better understanding the role of cAMP-PKA pathway in the formation of C. albicans biofilms.

Hannah Kim

Hannah Kim, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Ribosome Assembly in Saccharomyces cerevisiae: Investigating genetic interactions among Nop12, EBP2, and BRXI

Ribosomes are cellular components made up of 60s and 40s subunits that come together to act as protein factories inside living cells, catalyzing the reactions that join together individual amino acids to create polypeptides essential for the survival of that cell. The DNA of living cells codes for proteins that act as assembly factors that work in concert to assemble the ribosomes’ constituent parts: ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). These assembly factors have been the focus of intense research in recent years and over 200 assembly factors have been found to participate in the multi-faceted process of ribosome assembly. The goal of our research is to investigate the genetic interactions among three known assembly factors known to be involved in splicing of the 25s rRNA within 60s ribosomal subunit assembly: Nop12, EBP2 and BRXI. To this end, we made mutations wherein a selectable marker was integrated at the Nop12 site in either wild type or mutant BRXI or Ebp2 S. cerevisiae candidate strains. This method produced cold sensitive phenotypes in the Nop12 deleted mutants, and will allow for further genetic analysis of these double mutants.

David Markowitz

David Markowitz, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Mutant EcoRV as a Model System

Studies of the specific binding of the restriction endonuclease EcoRV to DNA can give valuable information on the specific binding of other proteins to DNA, such as polymerases and repressors. Most studies of EcoRV have been done using protein crystallization, but nuclear magnetic resonance (NMR) can give dynamic data because it is a solution phase technique. To this end we worked toward making mutant EcoRV protein that should not bind the metal ions necessary for cleavage of the DNA but should still bind the recognition sequence specifically. Such mutants would be perfect for NMR studies of EcoRV because they should remain bound to their recognition sequence instead of undergoing the extremely quick cleavage reaction and subsequently changing conformation. In the course of our research we were able to produce mutant genes for EcoRV through mutagenic PCR and subsequently insert some of those genes into final pET expression vectors for protein production. Studies of these mutant proteins could potentially provide information not only about the wild-type EcoRV but also about all proteins that bind specifically to DNA. Increased knowledge about such proteins could be useful across the field of biology (to improve PCR efficiency, gene therapy, etc.).

Korey Marshall

Korey Marshall, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Ribosome Assembly in Saccharomyces cerevisiae: Investigating genetic interactions among Nop12, EBP2, and BRXI

Ribosomes are cellular components made up of 60s and 40s subunits that come together to act as protein factories inside living cells, catalyzing the reactions that join together individual amino acids to create polypeptides essential for the survival of that cell. The DNA of living cells codes for proteins that act as assembly factors that work in concert to assemble the ribosomes’ constituent parts: ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). These assembly factors have been the focus of intense research in recent years and over 200 assembly factors have been found to participate in the multi-faceted process of ribosome assembly. The goal of our research is to investigate the genetic interactions among three known assembly factors known to be involved in splicing of the 25s rRNA within 60s ribosomal subunit assembly: Nop12, EBP2 and BRXI. To this end, we made mutations wherein a selectable marker was integrated at the Nop12 site in either wild type or mutant BRXI or Ebp2 S. cerevisiae candidate strains. This method produced cold sensitive phenotypes in the Nop12 deleted mutants, and will allow for further genetic analysis of these double mutants.

Kaitlyn Nowak

Kaitlyn Nowak, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Mutant EcoRV as a Model System

Studies of the specific binding of the restriction endonuclease EcoRV to DNA can give valuable information on the specific binding of other proteins to DNA, such as polymerases and repressors. Most studies of EcoRV have been done using protein crystallization, but nuclear magnetic resonance (NMR) can give dynamic data because it is a solution phase technique. To this end we worked toward making mutant EcoRV protein that should not bind the metal ions necessary for cleavage of the DNA but should still bind the recognition sequence specifically. Such mutants would be perfect for NMR studies of EcoRV because they should remain bound to their recognition sequence instead of undergoing the extremely quick cleavage reaction and subsequently changing conformation. In the course of our research we were able to produce mutant genes for EcoRV through mutagenic PCR and subsequently insert some of those genes into final pET expression vectors for protein production. Studies of these mutant proteins could potentially provide information not only about the wild-type EcoRV but also about all proteins that bind specifically to DNA. Increased knowledge about such proteins could be useful across the field of biology (to improve PCR efficiency, gene therapy, etc.).

Terrence Wong

Terrence Wong, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Required Domains of APC2 for Cortical Localization in Drosophila Melanogaster

Adenomatous polyposis coli (APC) is a protein found in higher eukaryotes. It functions as a negative regulator of Wnt signaling and it promotes stability of microtubules in the cytoskeleton. Too much Wnt signaling can cause cancer initiation and instability of the cytoskeleton can cause cancer progression. For this reason, APC acts as a tumor suppressor in humans and insufficient and/or dysfunctional APC is linked to over 80% of colon cancer cases. Drosophila melanogaster have two versions of APC, APC1 and APC2. It has been reported that APC2 localizes to the cortex of the cell through means of N-terminal Armadillo repeats and a novel C-terminal domain named C30 (McCartney et al. 2011). To study the specific residues in C30 that are required for cortical localization of APC2, we transfected Drosophila S2 cells with APC2 mutant constructs containing only the Armadillo repeats and subdomains of C30. Here, we show that only the Armadillo repeats and a subdomain of C30 are required for cortical localization of APC2. Within this subdomain of C30 lies a highly conserved region to many species of Drosophila. We hypothesize that this conserved region binds to the cortex through coiled-coil protein interactions. To disrupt coiled-coil formation, we propose a scheme in which hydrophobic residues found in the conserved region are changed to a short side-chain, polar amino acid such as threonine.