2010 SRI Participants-HHMI Undergraduate Program - Carnegie Mellon University

2010 Summer Research Institute (SRI) Participants

Catherine Byrd

Catherine Byrd, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Effects of Mutations in the Inositol Hexakisphosphate (IP6) Binding Region of hADAR2, an RNA Editing Enzyme

Adenosine deaminases that act on RNA (ADAR) are RNA editing enzymes that have been linked to proper neural function in animals. These enzymes catalyze a hydrolytic reaction that converts adenosine to inosine nucleotides in sections of RNA that are double stranded. ADAR has two cofactors in its catalytic domain: inositol hexakisphosphate (IP6) and a zinc ion. Both are required for ADAR to function and fold correctly. In this study, we created mutations in the catalytic domain of hADAR2 around IP6 using a site directed PCR mutagenesis technique with custom designed primers. pSc-ADAR plasmid containing the mutated hADAR2 gene was amplified and purified in Escherichia coli (E. coli). Then pSc-ADAR plasmid was transformed into Saccharomyces cerevisiae (S. cerevisiae) strain BCY123 for protein purification using galactose promoter induction. Nine mutations were made in the hADAR2 gene and six altered hADAR2 proteins were purified. These altered hADAR2 proteins can then be used for further structure function studies of hADAR2.

Danielle Devine

Danielle Devine, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Ribosomal Assembly in Saccharomyces cerevisiae: Interactions Between REA1, EBP2, ARX1, NOG2, and LSG1

Ribosomes are cellular components, consisting of 40s and 60s subunits, that are essential to life because they are responsible for the assembly of peptide chains via the process of translation. Mutations in ribosomal proteins and assembly factors have been linked to many diseases associated with an increased risk of cancer (Kressler et al). Research in this field over the past twenty years has uncovered over 200 assembly factors involved in the process, but little is known about the interactions of these factors during ribosome assembly. This project focuses specifically on pre-ribosomal particles during the export of the developing 60s subunit from the nucleus to the cytoplasm during ribosome assembly. Five genes, two with galactose inducible promoters (EBP2 and REA1) and three with Tandem Affinity Purification (TAP)-tags (ARX1, LSG1, and NOG2), were manipulated to study the interactions that take place during ribosomal biogenesis. We believe that arresting the transcription of Ebp2 and Rea1 using the galactose inducible promoters will disrupt ribosomal assembly in a way that will make the importance of Nog2, Lsg1, and Arx1 and their relations with Ebp2 and Rea1 apparent. To accomplish this, insertion of TAP tags via transformation and TAP tag purification were performed (Woolford et al). Overall, TAP tags were successfully inserted into the strains mutated with galactose inducible promoters and the pre-ribosomal complexes were isolated, allowing for analysis.

No Picture Available

Tiffany Ho, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Investigating Cleavage, Increasing Solubility, and Exploring Mg2+ Binding in EcoRV through Mutation Analysis

EcoRV is a Type II restriction endonuclease that cleaves the palindromic sequence GATATC. As do most restriction endonucleases, it requires Mg2+ or a closely related divalent metal ion to do so. Furthermore, EcoRV functions by linearly diffusing down a DNA strand it has loosely bound to until it reaches its recognition site for a subsequent cleavage. The overall goal is to increase EcoRV’s solubility by mutating certain amino acids on the enzyme’s surface to more polar amino acids, and to increase its reaction rate by mutating amino acids near its DNA binding sites to more positively-charged amino acids. Seventeen separate mutations, some on the enzyme’s surface and some near the ion binding site, were made using two different PCR mutagenesis techniques. One technique (Agilent Technologies) replicates the entire plasmid, resulting in the entire plasmid as a product, whereas the other technique (W. Kanoksilapatham,et. al. “Directed-Mutagenesis and Deletion Generated through an Improved Overlapping-Extension PCR Based Procedure.” Silpakorn U Science & Tech J Vol.1(2) page 8. 2007) creates a mutated gene that is subsequently amplified in later procedures. Both techniques were used to create mutated EcoRV products. Such an investigation of EcoRV ultimately helps better the understanding of the DNA restriction-modification system as well as the biochemical properties of EcoRV. So far, we have successfully created EcoRV-I62V and purified the mutated enzyme for functional analysis.

No Picture Available

Madison Kang, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Effects of Mutations in the Inositol Hexakisphosphate (IP6) Binding Region of hADAR2, an RNA Editing Enzyme

Adenosine deaminases that act on RNA (ADAR) are RNA editing enzymes that have been linked to proper neural function in animals. These enzymes catalyze a hydrolytic reaction that converts adenosine to inosine nucleotides in sections of RNA that are double stranded. ADAR has two cofactors in its catalytic domain: inositol hexakisphosphate (IP6) and a zinc ion. Both are required for ADAR to function and fold correctly. In this study, we created mutations in the catalytic domain of hADAR2 around IP6 using a site directed PCR mutagenesis technique with custom designed primers. pSc-ADAR plasmid containing the mutated hADAR2 gene was amplified and purified in Escherichia coli (E. coli). Then pSc-ADAR plasmid was transformed into Saccharomyces cerevisiae (S. cerevisiae) strain BCY123 for protein purification using galactose promoter induction. Nine mutations were made in the hADAR2 gene and six altered hADAR2 proteins were purified. These altered hADAR2 proteins can then be used for further structure function studies of hADAR2.

Linnea LaMon

Linnea LaMon, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Investigating Cleavage, Increasing Solubility, and Exploring Mg2+ Binding in EcoRV through Mutation Analysis

EcoRV is a Type II restriction endonuclease that cleaves the palindromic sequence GATATC. As do most restriction endonucleases, it requires Mg2+ or a closely related divalent metal ion to do so. Furthermore, EcoRV functions by linearly diffusing down a DNA strand it has loosely bound to until it reaches its recognition site for a subsequent cleavage. The overall goal is to increase EcoRV’s solubility by mutating certain amino acids on the enzyme’s surface to more polar amino acids, and to increase its reaction rate by mutating amino acids near its DNA binding sites to more positively-charged amino acids. Seventeen separate mutations, some on the enzyme’s surface and some near the ion binding site, were made using two different PCR mutagenesis techniques. One technique (Agilent Technologies) replicates the entire plasmid, resulting in the entire plasmid as a product, whereas the other technique (W. Kanoksilapatham,et. al. “Directed-Mutagenesis and Deletion Generated through an Improved Overlapping-Extension PCR Based Procedure.” Silpakorn U Science & Tech J Vol.1(2) page 8. 2007) creates a mutated gene that is subsequently amplified in later procedures. Both techniques were used to create mutated EcoRV products. Such an investigation of EcoRV ultimately helps better the understanding of the DNA restriction-modification system as well as the biochemical properties of EcoRV. So far, we have successfully created EcoRV-I62V and purified the mutated enzyme for functional analysis.

Katherine Lee

Katherine Lee, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Examining Pre-Ribosomal Complexes Through Tandem Affinity Purification

Ribosome assembly is a complicated process involving over 200 proteins and ribosomal RNAs. The purpose and goal of our research was to study the interactions of the selected proteins Nog2, Arx1, and Lsg1 with Rlp7 and Brx1 which are involved in ribosome assembly. Using Saccharomyces cerevisiae as our model organism, we focused on purifying the protein complexes containing the Nog2, Lsg1, and Arx1 assembly factors, all of which are associated with the 60s ribosomal subunit. Our goal was to study the interactions of these three proteins and RLP7 and BRX1 by tandem affinity purification (TAP), which can purify large protein complexes, tagged to the genes of interest in two strains: GAL-RLP7 and GAL-BRX1, both of which contain galactose inducible promoters. Therefore we are able to observe the interactions between ribosomal proteins by regulating the genes using either glucose or galactose. We succeeded in adding the TAP-tag to the NOG2 gene in the GAL-BRX1 and GAL-RLP7 strains, the ARX1 gene in the GAL-BRX1 and GAL-RLP7 strains, and the LSG1 gene in the GAL-BRX1 strain. We were able to verify correct integration of the TAP tag next to the gene of interest in the GAL-BRX1 strain with TAP-tagged NOG2. These strains will be used to look at the differences in the complexes purified depending on the activation of the RLP7 or BRX1 genes.

Andrew McCoy

Andrew McCoy, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Effects of Mutations in the Inositol Hexakisphosphate (IP6) Binding Region of hADAR2, an RNA Editing Enzyme

Adenosine deaminases that act on RNA (ADAR) are RNA editing enzymes that have been linked to proper neural function in animals. These enzymes catalyze a hydrolytic reaction that converts adenosine to inosine nucleotides in sections of RNA that are double stranded. ADAR has two cofactors in its catalytic domain: inositol hexakisphosphate (IP6) and a zinc ion. Both are required for ADAR to function and fold correctly. In this study, we created mutations in the catalytic domain of hADAR2 around IP6 using a site directed PCR mutagenesis technique with custom designed primers. pSc-ADAR plasmid containing the mutated hADAR2 gene was amplified and purified in Escherichia coli (E. coli). Then pSc-ADAR plasmid was transformed into Saccharomyces cerevisiae (S. cerevisiae) strain BCY123 for protein purification using galactose promoter induction. Nine mutations were made in the hADAR2 gene and six altered hADAR2 proteins were purified. These altered hADAR2 proteins can then be used for further structure function studies of hADAR2.

No Picture Available

Travis Nell, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Examining Pre-Ribosomal Complexes Through Tandem Affinity Purification

Ribosome assembly is a complicated process involving over 200 proteins and ribosomal RNAs. The purpose and goal of our research was to study the interactions of the selected proteins Nog2, Arx1, and Lsg1 with Rlp7 and Brx1 which are involved in ribosome assembly. Using Saccharomyces cerevisiae as our model organism, we focused on purifying the protein complexes containing the Nog2, Lsg1, and Arx1 assembly factors, all of which are associated with the 60s ribosomal subunit. Our goal was to study the interactions of these three proteins and RLP7 and BRX1 by tandem affinity purification (TAP), which can purify large protein complexes, tagged to the genes of interest in two strains: GAL-RLP7 and GAL-BRX1, both of which contain galactose inducible promoters. Therefore we are able to observe the interactions between ribosomal proteins by regulating the genes using either glucose or galactose. We succeeded in adding the TAP-tag to the NOG2 gene in the GAL-BRX1 and GAL-RLP7 strains, the ARX1 gene in the GAL-BRX1 and GAL-RLP7 strains, and the LSG1 gene in the GAL-BRX1 strain. We were able to verify correct integration of the TAP tag next to the gene of interest in the GAL-BRX1 strain with TAP-tagged NOG2. These strains will be used to look at the differences in the complexes purified depending on the activation of the RLP7 or BRX1 genes.

John Sadeghi

John Sadeghi, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Investigating Cleavage, Increasing Solubility, and Exploring Mg2+ Binding in EcoRV through Mutation Analysis

EcoRV is a Type II restriction endonuclease that cleaves the palindromic sequence GATATC. As do most restriction endonucleases, it requires Mg2+ or a closely related divalent metal ion to do so. Furthermore, EcoRV functions by linearly diffusing down a DNA strand it has loosely bound to until it reaches its recognition site for a subsequent cleavage. The overall goal is to increase EcoRV’s solubility by mutating certain amino acids on the enzyme’s surface to more polar amino acids, and to increase its reaction rate by mutating amino acids near its DNA binding sites to more positively-charged amino acids. Seventeen separate mutations, some on the enzyme’s surface and some near the ion binding site, were made using two different PCR mutagenesis techniques. One technique (Agilent Technologies) replicates the entire plasmid, resulting in the entire plasmid as a product, whereas the other technique (W. Kanoksilapatham,et. al. “Directed-Mutagenesis and Deletion Generated through an Improved Overlapping-Extension PCR Based Procedure.” Silpakorn U Science & Tech J Vol.1(2) page 8. 2007) creates a mutated gene that is subsequently amplified in later procedures. Both techniques were used to create mutated EcoRV products. Such an investigation of EcoRV ultimately helps better the understanding of the DNA restriction-modification system as well as the biochemical properties of EcoRV. So far, we have successfully created EcoRV-I62V and purified the mutated enzyme for functional analysis.

No Picture Available

Margaret Schervish, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Ribosomal Assembly in Saccharomyces cerevisiae: Interactions Between REA1, EBP2, ARX1, NOG2, and LSG1

Ribosomes are cellular components, consisting of 40s and 60s subunits, that are essential to life because they are responsible for the assembly of peptide chains via the process of translation. Mutations in ribosomal proteins and assembly factors have been linked to many diseases associated with an increased risk of cancer (Kressler et al). Research in this field over the past twenty years has uncovered over 200 assembly factors involved in the process, but little is known about the interactions of these factors during ribosome assembly. This project focuses specifically on pre-ribosomal particles during the export of the developing 60s subunit from the nucleus to the cytoplasm during ribosome assembly. Five genes, two with galactose inducible promoters (EBP2 and REA1) and three with Tandem Affinity Purification (TAP)-tags (ARX1, LSG1, and NOG2), were manipulated to study the interactions that take place during ribosomal biogenesis. We believe that arresting the transcription of Ebp2 and Rea1 using the galactose inducible promoters will disrupt ribosomal assembly in a way that will make the importance of Nog2, Lsg1, and Arx1 and their relations with Ebp2 and Rea1 apparent. To accomplish this, insertion of TAP tags via transformation and TAP tag purification were performed (Woolford et al). Overall, TAP tags were successfully inserted into the strains mutated with galactose inducible promoters and the pre-ribosomal complexes were isolated, allowing for analysis.

Kelly Shibuya

Kelly Shibuya, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Examining Pre-Ribosomal Complexes Through Tandem Affinity Purification

Ribosome assembly is a complicated process involving over 200 proteins and ribosomal RNAs. The purpose and goal of our research was to study the interactions of the selected proteins Nog2, Arx1, and Lsg1 with Rlp7 and Brx1 which are involved in ribosome assembly. Using Saccharomyces cerevisiae as our model organism, we focused on purifying the protein complexes containing the Nog2, Lsg1, and Arx1 assembly factors, all of which are associated with the 60s ribosomal subunit. Our goal was to study the interactions of these three proteins and RLP7 and BRX1 by tandem affinity purification (TAP), which can purify large protein complexes, tagged to the genes of interest in two strains: GAL-RLP7 and GAL-BRX1, both of which contain galactose inducible promoters. Therefore we are able to observe the interactions between ribosomal proteins by regulating the genes using either glucose or galactose. We succeeded in adding the TAP-tag to the NOG2 gene in the GAL-BRX1 and GAL-RLP7 strains, the ARX1 gene in the GAL-BRX1 and GAL-RLP7 strains, and the LSG1 gene in the GAL-BRX1 strain. We were able to verify correct integration of the TAP tag next to the gene of interest in the GAL-BRX1 strain with TAP-tagged NOG2. These strains will be used to look at the differences in the complexes purified depending on the activation of the RLP7 or BRX1 genes.

No Picture Available

Marianne Thaila, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Ribosomal Assembly in Saccharomyces cerevisiae: Interactions Between REA1, EBP2, ARX1, NOG2, and LSG1

Ribosomes are cellular components, consisting of 40s and 60s subunits, that are essential to life because they are responsible for the assembly of peptide chains via the process of translation. Mutations in ribosomal proteins and assembly factors have been linked to many diseases associated with an increased risk of cancer (Kressler et al). Research in this field over the past twenty years has uncovered over 200 assembly factors involved in the process, but little is known about the interactions of these factors during ribosome assembly. This project focuses specifically on pre-ribosomal particles during the export of the developing 60s subunit from the nucleus to the cytoplasm during ribosome assembly. Five genes, two with galactose inducible promoters (EBP2 and REA1) and three with Tandem Affinity Purification (TAP)-tags (ARX1, LSG1, and NOG2), were manipulated to study the interactions that take place during ribosomal biogenesis. We believe that arresting the transcription of Ebp2 and Rea1 using the galactose inducible promoters will disrupt ribosomal assembly in a way that will make the importance of Nog2, Lsg1, and Arx1 and their relations with Ebp2 and Rea1 apparent. To accomplish this, insertion of TAP tags via transformation and TAP tag purification were performed (Woolford et al). Overall, TAP tags were successfully inserted into the strains mutated with galactose inducible promoters and the pre-ribosomal complexes were isolated, allowing for analysis.