2012 SRI Participants-HHMI Undergraduate Program - Carnegie Mellon University

2012 Summer Research Institute (SRI) Participants

Hailey Brown

Hailey Brown, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Regulation of Endosomal Trafficking of 2-Adenergic receptor (B2AR)

The concentrations of Adrenergic receptors, in neuron cells, have been shown to play a major role in proper function of the sympathetic nervous system and the development of opioid drug addictions. Higher concentrations of B2AR will re-sensitize the cells to natural chemicals produced by our bodies, like endogenous opioids, so that a person becomes less dependent on external chemicals. Opioids can generally be found in the central and peripheral nervous system. Endosomal trafficking networks controlling the recycling and degradation of B2AR regulate the amount, and frequency, B2AR can be found at the cell surface. However, it is unknown what proteins allow the cell to differentiate between receptors bound for degradation and those bound for recycling.  It was hypothesized that a theoretical “protein x” binds selectively to a phosphorylated adrenergic receptor and causes it to recycle. In this experiment, three mutations were induced through PCR mutagenesis, in the beta-2 adrenergic receptor (B2AR) in an attempt to identify the protein mechanisms regulating endosomal trafficking of this receptor. It was discovered that the effects of receptor phosphorylation could be simulated by mutation of two codons on the receptor tail DNA sequence. Two of the mutations were done at these two codons (the phosphorylation kinase A (PKA) site) to look at the effect of phosphorylation on protein binding affinity. The other mutation, located downstream of the PKA site, is thought to restrict the recycling pathway and its mutated version allows for rapid recycling. With our in vitro proteins, along with the GST tag, that came from the pGEX vector, this will allow us to see which human proteins bind to this tag.  This can lead to the identification of our theoretical protein x, which may play a role in controlling the recycling of B2AR and similar cell receptors.
Dagney Cooke

Dagney Cooke, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

The Origin of Unique Ribose Biosynthesis in Yeast

Shb17, a gene from Saccharomyces cerevisiae, provides an alternative route to ribose production under low oxygen conditions. Genes similar to Shb17 have been found in Agrobacterium and Candida albicans, but not in more closely related families such as plants or animals, suggesting horizontal gene transfer from Agrobacterium to yeast. To test the possibility of horizontal gene transfer comparisons will be made between the metabolic function of Shb17-like genes found in C. albicans and Agrobacterium, and the Shb17 gene found in S. cerevisiae. The three Shb17-like genes were inserted into triple knockout yeast strains to test whether these proteins restore wildtype growth. Triple knockout strains refer to yeast strains that have multiple deletions of genes essential for ribose production; in this case, the genes include zwf1, tal1 and Shb17. This mutation results in inhibited growth. The transformation of Shb17 into the triple knockout strain restores wildtype growth. Gateway® Technology was used to provide a fast and efficient way to move synthetic DNA sequences into a vector system appropriate for plasmid amplification in E. coli and gene expression in S. cerevisiae. Growth curves compare the growth rates of triple knockout yeast transformed with Shb17-like genes to see if they restore wildtype growth. Sequence alignments identified an additional loop in the Shb17 gene that is unique to Shb17 and not present in the Shb17 like-genes in other organisms. Consequently, a mutation of the Shb17 gene to delete this loop. Transforming the mutant gene into triple knockout S. cerevisiae and comparing the growth rate to the same yeast strain transformed with Shb17 will indicate whether the mutant still performs the same metabolic function. This will give insight as to whether the loop structure essential to Shb17’s function in S. cerevisiae.

Sean Kane

Sean Kane, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Regulation of Endosomal Trafficking of 2-Adenergic receptor (B2AR)

The concentrations of Adrenergic receptors, in neuron cells, have been shown to play a major role in proper function of the sympathetic nervous system and the development of opioid drug addictions. Higher concentrations of B2AR will re-sensitize the cells to natural chemicals produced by our bodies, like endogenous opioids, so that a person becomes less dependent on external chemicals. Opioids can generally be found in the central and peripheral nervous system. Endosomal trafficking networks controlling the recycling and degradation of B2AR regulate the amount, and frequency, B2AR can be found at the cell surface. However, it is unknown what proteins allow the cell to differentiate between receptors bound for degradation and those bound for recycling.  It was hypothesized that a theoretical “protein x” binds selectively to a phosphorylated adrenergic receptor and causes it to recycle. In this experiment, three mutations were induced through PCR mutagenesis, in the beta-2 adrenergic receptor (B2AR) in an attempt to identify the protein mechanisms regulating endosomal trafficking of this receptor. It was discovered that the effects of receptor phosphorylation could be simulated by mutation of two codons on the receptor tail DNA sequence. Two of the mutations were done at these two codons (the phosphorylation kinase A (PKA) site) to look at the effect of phosphorylation on protein binding affinity. The other mutation, located downstream of the PKA site, is thought to restrict the recycling pathway and its mutated version allows for rapid recycling. With our in vitro proteins, along with the GST tag, that came from the pGEX vector, this will allow us to see which human proteins bind to this tag.  This can lead to the identification of our theoretical protein x, which may play a role in controlling the recycling of B2AR and similar cell receptors.
Kacey Idouchi

Kacey Idouchi, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Regulation of Endosomal Trafficking of 2-Adenergic receptor (B2AR)

The concentrations of Adrenergic receptors, in neuron cells, have been shown to play a major role in proper function of the sympathetic nervous system and the development of opioid drug addictions. Higher concentrations of B2AR will re-sensitize the cells to natural chemicals produced by our bodies, like endogenous opioids, so that a person becomes less dependent on external chemicals. Opioids can generally be found in the central and peripheral nervous system. Endosomal trafficking networks controlling the recycling and degradation of B2AR regulate the amount, and frequency, B2AR can be found at the cell surface. However, it is unknown what proteins allow the cell to differentiate between receptors bound for degradation and those bound for recycling.  It was hypothesized that a theoretical “protein x” binds selectively to a phosphorylated adrenergic receptor and causes it to recycle. In this experiment, three mutations were induced through PCR mutagenesis, in the beta-2 adrenergic receptor (B2AR) in an attempt to identify the protein mechanisms regulating endosomal trafficking of this receptor. It was discovered that the effects of receptor phosphorylation could be simulated by mutation of two codons on the receptor tail DNA sequence. Two of the mutations were done at these two codons (the phosphorylation kinase A (PKA) site) to look at the effect of phosphorylation on protein binding affinity. The other mutation, located downstream of the PKA site, is thought to restrict the recycling pathway and its mutated version allows for rapid recycling. With our in vitro proteins, along with the GST tag, that came from the pGEX vector, this will allow us to see which human proteins bind to this tag.  This can lead to the identification of our theoretical protein x, which may play a role in controlling the recycling of B2AR and similar cell receptors.

Annette Ko

Annette Ko, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Investigating the Regenerative Process in Patiria miniata Larvae

Regeneration, defined as the process by which some organisms replace or restore lost or amputated body parts, has been widely observed across the Metazoa. Specifically, echinoderms belonging to the class Asteroidea (sea stars) are able to undergo complete organogenesis in both their larval and adult forms. Furthermore, sea stars are known to be deuterostomes and share their embryonic development patterns with humans. This similarity makes sea stars more closely related to vertebrates than other invertebrates, and thus they may provide insight into regeneration in humans. While literature describing sea star regeneration exists, specific characteristics, such as gene expression throughout the regenerative process and the up- or down- regulation of genes, are not known. In order to better characterize this process in sea stars, larvae of the species Patiria miniata were bisected into anterior and posterior segments, and the organisms’ regeneration was observed over time. A time series suggested that a larvae's ability to regenerate its lost half was largely independent of the age of the larvae pre-bisection.  In situ hybridization was then performed utilizing mRNA probes targeting the genes OneCut (ciliary bands), FoxA (gut outgrowth), and Vasa (coeloms). It was determined that all cell types of interest were regenerated in the expected morphological regions. A cDNA library was generated from the RNA of bisected larvae and qPCR was used to further refine and quantify the up- and down- regulation of the genes of interest. Our results contribute to an initial framework for understanding the regenerative process and its associated genetic changes in sea stars.

Lazar LaLone

Lazar LaLone, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Investigating the Regenerative Process in Patiria miniata Larvae

Regeneration, defined as the process by which some organisms replace or restore lost or amputated body parts, has been widely observed across the Metazoa. Specifically, echinoderms belonging to the class Asteroidea (sea stars) are able to undergo complete organogenesis in both their larval and adult forms. Furthermore, sea stars are known to be deuterostomes and share their embryonic development patterns with humans. This similarity makes sea stars more closely related to vertebrates than other invertebrates, and thus they may provide insight into regeneration in humans. While literature describing sea star regeneration exists, specific characteristics, such as gene expression throughout the regenerative process and the up- or down- regulation of genes, are not known. In order to better characterize this process in sea stars, larvae of the species Patiria miniata were bisected into anterior and posterior segments, and the organisms’ regeneration was observed over time. A time series suggested that a larvae's ability to regenerate its lost half was largely independent of the age of the larvae pre-bisection.  In situ hybridization was then performed utilizing mRNA probes targeting the genes OneCut (ciliary bands), FoxA (gut outgrowth), and Vasa (coeloms). It was determined that all cell types of interest were regenerated in the expected morphological regions. A cDNA library was generated from the RNA of bisected larvae and qPCR was used to further refine and quantify the up- and down- regulation of the genes of interest. Our results contribute to an initial framework for understanding the regenerative process and its associated genetic changes in sea stars.
Gordon Pherribo

Gordon Pherribo, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

The Origin of Unique Ribose Biosynthesis in Yeast

Shb17, a gene from Saccharomyces cerevisiae, provides an alternative route to ribose production under low oxygen conditions. Genes similar to Shb17 have been found in Agrobacterium and Candida albicans, but not in more closely related families such as plants or animals, suggesting horizontal gene transfer from Agrobacterium to yeast. To test the possibility of horizontal gene transfer comparisons will be made between the metabolic function of Shb17-like genes found in C. albicans and Agrobacterium, and the Shb17 gene found in S. cerevisiae. The three Shb17-like genes were inserted into triple knockout yeast strains to test whether these proteins restore wildtype growth. Triple knockout strains refer to yeast strains that have multiple deletions of genes essential for ribose production; in this case, the genes include zwf1, tal1 and Shb17. This mutation results in inhibited growth. The transformation of Shb17 into the triple knockout strain restores wildtype growth. Gateway® Technology was used to provide a fast and efficient way to move synthetic DNA sequences into a vector system appropriate for plasmid amplification in E. coli and gene expression in S. cerevisiae. Growth curves compare the growth rates of triple knockout yeast transformed with Shb17-like genes to see if they restore wildtype growth. Sequence alignments identified an additional loop in the Shb17 gene that is unique to Shb17 and not present in the Shb17 like-genes in other organisms. Consequently, a mutation of the Shb17 gene to delete this loop. Transforming the mutant gene into triple knockout S. cerevisiae and comparing the growth rate to the same yeast strain transformed with Shb17 will indicate whether the mutant still performs the same metabolic function. This will give insight as to whether the loop structure essential to Shb17’s function in S. cerevisiae.
Daniele Sori

Daniele Sori, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

The Origin of Unique Ribose Biosynthesis in Yeast

Shb17, a gene from Saccharomyces cerevisiae, provides an alternative route to ribose production under low oxygen conditions. Genes similar to Shb17 have been found in Agrobacterium and Candida albicans, but not in more closely related families such as plants or animals, suggesting horizontal gene transfer from Agrobacterium to yeast. To test the possibility of horizontal gene transfer comparisons will be made between the metabolic function of Shb17-like genes found in C. albicans and Agrobacterium, and the Shb17 gene found in S. cerevisiae. The three Shb17-like genes were inserted into triple knockout yeast strains to test whether these proteins restore wildtype growth. Triple knockout strains refer to yeast strains that have multiple deletions of genes essential for ribose production; in this case, the genes include zwf1, tal1 and Shb17. This mutation results in inhibited growth. The transformation of Shb17 into the triple knockout strain restores wildtype growth. Gateway® Technology was used to provide a fast and efficient way to move synthetic DNA sequences into a vector system appropriate for plasmid amplification in E. coli and gene expression in S. cerevisiae. Growth curves compare the growth rates of triple knockout yeast transformed with Shb17-like genes to see if they restore wildtype growth. Sequence alignments identified an additional loop in the Shb17 gene that is unique to Shb17 and not present in the Shb17 like-genes in other organisms. Consequently, a mutation of the Shb17 gene to delete this loop. Transforming the mutant gene into triple knockout S. cerevisiae and comparing the growth rate to the same yeast strain transformed with Shb17 will indicate whether the mutant still performs the same metabolic function. This will give insight as to whether the loop structure essential to Shb17’s function in S. cerevisiae.
Sowmya Yennam

Sowmya Yennam, Carnegie Mellon University

Mentor: Dr. Gordon Rule and Dr. Maggie Braun

Investigating the Regenerative Process in Patiria miniata Larvae

Regeneration, defined as the process by which some organisms replace or restore lost or amputated body parts, has been widely observed across the Metazoa. Specifically, echinoderms belonging to the class Asteroidea (sea stars) are able to undergo complete organogenesis in both their larval and adult forms. Furthermore, sea stars are known to be deuterostomes and share their embryonic development patterns with humans. This similarity makes sea stars more closely related to vertebrates than other invertebrates, and thus they may provide insight into regeneration in humans. While literature describing sea star regeneration exists, specific characteristics, such as gene expression throughout the regenerative process and the up- or down- regulation of genes, are not known. In order to better characterize this process in sea stars, larvae of the species Patiria miniata were bisected into anterior and posterior segments, and the organisms’ regeneration was observed over time. A time series suggested that a larvae's ability to regenerate its lost half was largely independent of the age of the larvae pre-bisection.  In situ hybridization was then performed utilizing mRNA probes targeting the genes OneCut (ciliary bands), FoxA (gut outgrowth), and Vasa (coeloms). It was determined that all cell types of interest were regenerated in the expected morphological regions. A cDNA library was generated from the RNA of bisected larvae and qPCR was used to further refine and quantify the up- and down- regulation of the genes of interest. Our results contribute to an initial framework for understanding the regenerative process and its associated genetic changes in sea stars.