Click here for the research poster. Please download the file.

Melissa Reeves, University of California, San Francisco

"Exploring the Impact of Speckled Clonal Patterning in Skin Carcinomas"

Abstract:

Tumors are growths of evolving, genetically diverse populations that typically arise from a single mutant cell. As this mutant cell proliferates, the cells stemming from it progressively pick up more mutations, creating genetic heterogeneity, a characteristic which has been linked with worse prognosis and increased treatment difficulty. In order to study tumor heterogeneity, my lab utilizes a lineage tracing system that labels clonal populations with distinct fluorescent reporters to track clonal evolution throughout tumor progression of squamous cell carcinomas. While analyzing tumors generated using this system, I have observed that approximately one-third of them display a “speckling” pattern wherein these malignant tumors have rare, non-clustered cells from a minor clone “speckled” throughout the dominant clone(s). At present, the role of these minor populations in malignancy is poorly understood. To assess the dynamics that lead to these minor clones, I am comparing the proliferation, migration, and competitive fitness of the major and minor populations of one speckled tumor, 1237A. I have thus far established that the major and minor clones of 1237A grow at the same rate individually in mice, suggesting a similar level of fitness. My in vitro wound closure experiments have additionally shown that these clones close wounds at the same rate, indicating a similar level of migratory ability. Furthermore, I am conducting experiments in which these clones are grown together, both in cell culture and in vivo, to test for evidence of competition and if the original speckled pattern is consistently recapitulated in tumors. In culmination, these experiments will illuminate the behavior of minor clonal populations that are often overlooked due to their rarity within individual tumors and ultimately determine their role in tumor development.

Click here for the research poster. Click here for the video presentation.

Kathleen Collins, Department of Molecular and Cell Biology

"Specificity of Binding of Oryzias latipes R2 Retrotransposon Protein to Target 28S rDNA"

Abstract:

R2 is a family of eukaryotic non-long terminal repeat retrotransposable elements that insert into a conserved sequence within the 28S ribosomal RNA gene (28S rDNA). R2 elements integrate site-specifically using a mechanism that incurs minimal DNA damage, suggesting their potential in gene therapy. While R2 is sequence-specific, the protein domains responsible for specificity are unknown. Here, we hypothesized that the R2 N-terminal domain (NTD), which contains DNA-binding motifs, specifically targets 28S rDNA. The NTD of Oryzias latipes R2, a representative ancestral clade protein, was purified and assayed by electrophoretic mobility shift for its binding to on- and off-target sequences. The NTD was observed to bind a 64 bp 28S rDNA sequence and preferentially target DNA upstream of the insertion site. Furthermore, the NTD was found to be specific for its 28S rDNA target compared to a competing 18S rDNA sequence. This implies that the NTD is sufficient to confer DNA-binding specificity to the O. latipes R2 protein and may partly explain distinct targeting between R2 and related 18S rDNA-specific retroelements.

Click here for the poster.

Dave Savage, Department of Molecular & Cell Biology, University of California, Berkeley

"Evaluating the Mutation Space of R. rubrum RuBisCO"

Abstract:

The Calvin Benson Bassham (CBB) cycle is the carbon-fixing pathway in photosynthesis. Rubisco is a key enzyme in the CBB cycle and is responsible for converting atmospheric CO2 into biomass. Rubisco is the most abundant enzyme on the planet, accounting for as much as 50% of the soluble protein of some plants. The role of Rubisco in carbon sequestration makes it a key enzyme for conservation and climate change study. This project focuses on a form II Rhodospirillum rubrum rubisco, a simple dimeric protein. Though analysis of a few select mutations on this Rubisco have been conducted, no current studies or set of studies quantify the effect of mutations at every site of the linked dimer. Due to its homodimeric nature, any genetic mutations in the rbcL gene results in mutations at two locations in the functional dimer. Therefore, in order to better explore the mutation space, the rbcL gene was duplicated into an expression plasmid separated by a linker using Gibson assembly. The modified plasmid was transformed into a specially engineered E. coli strain (CCMB1) made by the Savage lab whose unique dependence on Rubisco links Rubisco function directly with growth, making it possible to identify fixation rates of the enzyme directly through growth assays. We determined the viability of a linked double mutant using a 18 bp linker sequence, and showed functional single active site K191A mutants both in vitro and in vivo. Normal dimer assembly was validated by native gel and size exclusion chromatography. Thus, by showing activity of the linked R. rubrum form II dimer and of single site mutants we show linked rubisco biochemistry as a viable malleable mutation space for simple form II rubisco. A Library was created to establish a full mutation set, and analysis is underway to explore the local sequence landscape of this enzyme and the effect of symmetry on that landscape. This analysis will help us better understand the full malleable mutation space of R. rubrum Rubisco.

Click here for the research video.

Marla Feller, Department of Molecular & Cell Biology

"Intrinsically Photosensitive Retinal Ganglion Cell Participation in Spontaneous and Light Evoked Activity in the Developing Mouse"

Abstract:

When exposed to bright light, neonatal mice pups will exhibit photoaversive behavior by turning away from the light source. This behavior is mediated by intrinsically photosensitive Retinal Ganglion Cells (ipRGCs), a class of output neurons of the retina which express the light-sensitive protein Melanopsin.

However, it is also known that during this period of development, retinal ganglion cells are actively involved in spontaneous activity patterns known as retinal waves, and it was thought that all retinal ganglion cells including ipRGCs participated in waves.

This raises the question of how ipRGCs accurately encode outside light level and mediate light aversive behavior, when in the presence of retinal waves?

To answer this question, I analyzed calcium imaging footage of waves on acutely isolated and flattened retinas from mice of postnatal day 6-9. I made wave-triggered averages of individual light-responsive cells in these fields of views in order to find whether they participate in waves. When I classified the cells according to their light response and compared their wave triggered averages, I found that the cluster with high sustained light responses uniquely contained a high proportion of cells that did not participate in waves. This functional cluster corresponds to the anatomical ipRGC subtype known to mediate this photoaversive behavior in mice pups.

This high proportion of high sustained ipRGCs that are exempt from waves explains how these cells can faithfully encode light intensity and mediate light aversive behavior, despite the presence of retinal waves.

Click here for the research video.

Jay Keasling, Joint Bioenergy Institute, Lawrence Berkeley National Laboratory

"Engineering the Chlorinated Kavalactone Biosynthetic Pathway into Psuedomonas putida"

Abstract:

Kavalactones are a class of psychoactive plant natural products derived from the kava plant, Piper methysticum. Kava has known anxiolytic effects and is widely used by indigenous cultures of the South Pacific as a ritualistic medicine. Recent scientific research into the potential applications of kavalactones for pharmaceuticals has shown promising results, however kavalactones derived directly from the plant also contain toxic compounds. Producing kavalactones in Pseudomonas putida therefore presents an alternative production strategy that will allow us to have better control over the product profile and to create novel compounds. Halogenation of small molecules have been found to have interesting pharmacological properties including modulation of human cell receptors and cytotoxicity. In this project, we present an experimental design to engineer a chlorinated kavalactone pathway into P. putida. Using DNA shuffling techniques with two enzymes of interest, we will create an engineered enzyme. This enzyme will then be introduced as the final step in our existing biosynthetic kavalactone pathway and then screened for activity using mass spectrometry. Additionally, we propose an alternative mutagenesis strategy informed by structural alignment for creation of an engineered halogenase. Finally, we plan to employ preparatory LC and biochemical assays to purify and assess the activity of these novel products.

Click here for research video. Click here for the slides.

Charles J. Limb, University of California, San Francisco, Parnassus

"Analyzing Pitch and Timbre Interaction in Cochlear Implant Users"

Abstract:

Sensorineural hearing loss requires a cochlear implant in order to hear. Cochlear implant users regain decent speech perception, but struggle with music perception. Many studies of CI user music perception typically involve analyzing single aspects of music perception (ex. just rhythm). However, music is perceived as a mixture of multiple aspects. To further understand complex music perception in CI users, it is crucial to understand how these aspects affect each other when perceived—the interaction of these aspects. Based on previous studies, normal-hearing listeners perceive pitch—the frequency of a sound—and timbre—the type of instrument—with this interaction. The question now is if this interaction also exists for cochlear implant users. This online project focused on a small part of the interaction and compared cochlear implant users and normal-hearing listeners. Participants took a remote, online survey through UCSF Qualtrics in which they listened to sound files of a trumpet and a violin playing different pitches. They are asked to sort these sounds based on their pitch and/or timbre. Control tasks involved sorting stimuli varying in one attribute; experimental tasks involved sorting stimuli varying in both attributes. Reaction times were recorded for each question. We hypothesized that both populations would show facilitation in the experimental task—shorter reaction times in experimental tasks compared to control. CI users took significantly longer than NH listeners, indicating they are processing these attributes slower. Facilitation was found in the CI population, but not in NH listeners. This finding in NH listeners may be due to the task being too easy or participants focusing on only the easiest-to-perceive attribute in the experimental tasks. Error rates in CI users support the reaction time finding, but it is unclear if this is due to CI users focusing on the easiest-to-perceive attribute or actually attending to both stimuli at the same time. Further, more rigorous testing is needed to parse out this actual interaction.

Click here for the research poster.

Jay D. Keasling, Joint BioEnergy Institute

"Computational Methods for Enhancing Isoprenol Production in Pseudomonas putida"

Abstract:

Pseudomonas putida is an emerging microbial chassis for metabolic engineering applications due to its well characterized central metabolism, ability to catabolize a broad range of aliphatic and aromatic carbon sources, robustness to physicochemical stress, and amenability to genetic modifications. In this project, the design of methods to increase production of the drop-in biofuel isoprenol were computationally analyzed using the genome-scale metabolic reconstruction of Pseudomonas putida, iJN1462. The design and analysis of metabolic engineering strategies in silico can guide future experiments for improving the production of isoprenol to reach titers and yields necessary to become a viable fossil fuel alternative.

Click here for the pdf

Dr. Felix Feng , University of California, San Francisco

"Determining the Impact of PARP Inhibitors on Molecular Subtypes of Aggressive Prostate Cancer"

Abstract:

The genomic landscape of metastatic castration-resistant prostate cancer (mCRPC) is very diverse, with many uncharacterized molecular subtypes. Poly (ADP-ribose) polymerase (PARP) inhibitors are effective against mCRPCs characterized by mutations in BRCA1 and BRCA2. However, the efficacy of PARP inhibitors on tumors marked by a loss-of-function mutation in cyclin-dependent kinase 12 (CDK12) or other genes in the homologous recombination pathway is unclear. Here we used CRISPR-Cas9 to knockout CDK12 and knockdown BARD1 and BRIP1 and explored the use of PARP inhibitors as a treatment for aggressive prostate cancers. Simultaneously, we analyzed the impact of CDK12 loss on gene expression and downstream pathways. We found that CDK12 controls the expression of several homologous recombination genes and downregulated the E2F targets and G2/M checkpoint. Furthermore, mutations in CDK12, BARD1, or BRIP1 conferred sensitivity to PARP inhibitors. In conclusion, exploring new genomic subtypes for PARP inhibitor therapy and developing computational tools for genomic analysis provides new insights for precision oncology.

Click here for an interactive website. Click here for information about a downloadable Python package. Click here for the research poster.

John Kuriyan, Department of Molecular & Cell Biology, University of California, Berkeley

"Decrease of Stability in Ras Mutants Can Induce Activation"

Abstract:

Ras is a critical G-protein which acts as a molecular switch, cycling between a GTP-bound ON state and GDP-bound OFF state. Ras notably participates in the MAP Kinase pathway resulting in cell proliferation and growth when GTP-bound Ras activates the Raf kinase. Gain of function (GOF) Ras mutations are commonly found in human cancers, in particular at positions G12, G13, and Q61, with distinct structural and biochemical properties. To better understand the role of each mutation in Ras, prior work in the Kuriyan Lab included the adaptation of a bacterial 2 hybrid (B2H) assay, where Ras mutations were screened by examining their effect on the binding of an effector protein. The phenotypic screen of Ras showed that there are additional GOF mutations besides the ones found in cancer. The B2H screen was conducted in both long (residues 1-180) and short (residues 2-166), truncated versions of Ras, generating two next generation sequencing (NGS) datasets. In this work, I present results from a pulse proteolysis assay in the long (residues 1-173) and short (residues 1-166) Ras constructs and validate pulse proteolysis as an assay to measure the change in protein stability corresponding to the phenotypic screen results in GOF Ras variants. I also present my work in developing computational tools for the processing, analysis, and visualization of NGS datasets.

Click here for the poster.

Dr. John Kuriyan, Department of Molecular & Cell Biology

"Investigation of Conserved Glutamate Residue in the nRTK Family"

Abstract:

Non-receptor tyrosine kinases (nRTKs) are a sub-group of tyrosine kinases, which can regulate diverse cellular functions such as cell survival, division, gene expression, immune response, etc. They have a shared kinase domain and many other domains such as SH2, SH3 which are protein-protein interacting domains. There exists a highly conserved glutamate residue outside the activation loop region of this superfamily. Previous studies have shown that substitution of this glutamate residue by glycine in the protein kinases SRC (E378G) and BTK (E513G) yields highly active kinases with transforming potential in cells. Furthermore, in BTK, glutamate to glycine mutation in conjunction with a gatekeeper threonine residue mutation confers resistance to inhibitors targeting the kinase activation site. This project aims to develop a high throughput cell-based assay by constructing a deep mutagenesis library of this conserved position and using next generation sequencing to evaluate SRC, BTK and ABL (E355G, Abl 1a numbering) mutants under the selective pressure of IL-3 presence/absence.

Click here for research poster.

Evangelina Nogales, Department of Molecular & Cell Biology

"The Structural Characterization of the SIR Complex"

Abstract:

Precise regulation of gene expression is necessary for all cellular processes and involves transcriptional activation, repression, and homeostasis that is achieved by highly specific protein complexes. My research focuses on a yeast protein complex, the SIR complex, which is involved in heterochromatin, or silent chromatin, formation. Silent chromatin is a highly compact DNA structure that is inaccessible to transcription machinery so the genes can’t be expressed. Through comparative analyses of the different mechanisms of transcription repression processes between humans and S. cerevisiae, we will be able to gain a better understanding of how deregulation of SIR proteins and their conserved human homologs (Sirtuins) may play a role in diseases like diabetes and Alzheimer’s. Previous work investigating the mechanism of heterochromatin formation by the SIR complex has primarily involved individual domains of these proteins and not the entire functional complex and how the intact complex interacts with nucleosomes. The aim of my work is to gain a deeper understanding of how the SIR complex functions as a whole. Since the mechanism of recruitment of the SIR complex to specific genes has not yet been determined, I will investigate this mechanism using cryo-electron microscopy to image the SIR-nucleosome interactions. One of the questions I will aim to answer is whether the catalytic deacetylase activity and the formation of heterochromatin are two distinct activities of the complex or whether deacetylation is a required step for heterochromatin formation. My research aims to provide mechanistic insights into the regulation of transcription by multi-component protein assemblies.