Biology Commons^™

Chapter 26 – Multiple Color Single Molecule Tirf Imaging And Tracking Of Maps And Motors, Jennifer L. Ross, Ram Dixit

Biology Faculty Research

Microtubules are part of a complex mechano-chemical network inside cells. In order to understand how the components of these systems work together, careful in vitro experiments must be performed with added complexity. These experiments can ideally image all the interacting species. In order to image these molecules, multiple-color fluorescence imaging can be performed. In this chapter, we describe some methods for performing multiple-color single molecule fluorescence imaging using total internal reflection fluorescence microscopy. We give several specific examples of species of microtubule-associate proteins and motors that can be examined with detailed protocols for labeling, purification, and imaging.

A Three-Dimensional Computer Simulation Model Reveals The Mechanisms For Self-Organization Of Plant Cortical Microtubules Into Oblique Arrays, Ezgi Can Eren, Ram Dixit, Natarajan Gautam

Biology Faculty Research

The noncentrosomal cortical microtubules (CMTs) of plant cells self-organize into a parallel three-dimensional (3D) array that is oriented transverse to the cell elongation axis in wild-type plants and is oblique in some of the mutants that show twisted growth. To study the mechanisms of CMT array organization, we developed a 3D computer simulation model based on experimentally observed properties of CMTs. Our computer model accurately mimics transverse array organization and other fundamental properties of CMTs observed in rapidly elongating wild-type cells as well as the defective CMT phenotypes observed in the Arabidopsis mor1-1 and fra2 mutants. We found that CMT …

Drosophila Risc Component Vig And Its Homolog Vig2 Impact Heterochromatin Formation, Elena Gracheva, Monica Dus, Sarah C.R. Elgin

Biology Faculty Research

Heterochromatin formation plays an important role in gene regulation and the maintenance of genome integrity. Here we present results from a study of the D. melanogaster gene vig, encoding an RNAi complex component and its homolog vig2 (CG11844) that support their involvement in heterochromatin formation and/or maintenance. Protein null mutations vig(EP812) and vig2(PL470) act as modifiers of Position Effect Variegation (PEV). VIG and Vig2 are present in polytene chromosomes and partially overlap with HP1. Quantitative immunoblots show depletion of HP1 and HP2 (large isoform) in isolated nuclei from the vig(EP812) mutant. The vig2(PL470) mutant strain demonstrates a decreased level of …

Unlocking The Secrets Of The Genome, Susan Celniker, Laura Dillon, Mark Gerstein, Kristin Gunsalus, Steven Henikoff, Gary Karpen, Manolis Kellis, Eric Lai, Jason Lieb, David Macalpine, Gos Micklem, Fabio Piano, Michael Snyder, Lincoln Stein, Kevin White, Robert Waterston, Sarah C.R. Elgin

Biology Faculty Research

Despite the successes of genomics, little is known about how genetic information produces complex organisms. A look at the crucial functional elements of fly and worm genomes could change that.

A Lot About A Little Dot - Lessons Learned From Drosophila Melanogaster Chromosome 4, Nicole Riddle, Christopher Shaffer, Sarah C.R. Elgin

Biology Faculty Research

The fourth chromosome of Drosophila melanogaster has a number of unique properties that make it a convenient model for the study of chromatin structure. Only 4.2 Mb overall, the 1.2 Mb distal arm of chromosome 4 seen in polytene chromosomes combines characteristics of heterochromatin and euchromatin. This domain has a repeat density of ~35%, comparable to some pericentric chromosome regions, while maintaining a gene density similar to that of the other euchromatic chromosome arms. Studies of position-effect variegation have revealed that heterochromatic and euchromatic domains are interspersed on chromosome 4, and both cytological and biochemical studies have demonstrated that chromosome …

Small Rna-Directed Heterochromatin Formation In The Context Of Development: What Flies Might Learn From Fission Yeast, Kathryn Huisinga, Sarah C.R. Elgin

Biology Faculty Research

A link between the RNAi system and heterochromatin formation has been established in several model organisms including Schizosaccharomyces pombe and Arabidopsis thaliana. However, the data to support a role for small RNAs and the associated machinery in transcriptional gene silencing in animal systems is more tenuous. Using the S. pombe system as a model, we analyze the role of small RNA pathway components and associated small RNAs in regulating transposable elements and potentially directing heterochromatin formation at these elements in Drosophila melanogaster.

Coiled-Coil-Mediated Dimerization Is Not Required For Myosin Vi To Stabilize Actin During Spermatid Individualization In Drosophila Melanogaster, Tatsuhiko Nogushi, Deborah J. Frank, Mamiko Isaji, Kathryn G. Miller

Biology Faculty Research

Myosin VI is a pointed-end-directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function …

Undergraduate Research. Genomics Education Partnership, D Lopatto, C Alvarez, D Barnard, C Chandrasekaran, H-M Chung, C Du, T Eckdahl, A Goodman, C Hauser, C Jones, O Kopp, G Kuleck, G Mcneil, R Morris, J Myka, A Nagengast, P Overvoorde, J Poet, K Reed, G Regisford, D Revie, A Rosenwald, K Saville, M Shaw, G Skuse, C Smith, M Smith, M Spratt, J Stamm, J Thompson, B Wilson, C Witkowski, J Youngblom, Wilson Leung, Christopher Shaffer, Jeremy Buhler, Elaine Mardis, Sarah C.R. Elgin

Biology Faculty Research

The Genomics Education Partnership offers an inclusive model for undergraduate research experiences incorporated into the academic year science curriculum, with students pooling their work to contribute to international data bases.

Identifying And Characterizing Arabidopsis Jasmonate Signaling Components Manipulated By Pseudomonas Syringae, Neva Laurie-Berry

Arts & Sciences Graduate Student Theses and Dissertations

Jasmonate signaling is critical for susceptibility of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae, as demonstrated by the reduced susceptibility of coil mutant plants. The infection responses of another jasmonate-insensitive mutant, jinl, had previously been unknown. This work demonstrates that susceptibility to P. syringae requires activity of the transcription factor JTN1/MYC2. Reduced susceptibility correlates with reduced sensitivity to the P. syringae phytotoxin coronatine, a molecular mimic of the endogenous jasmonate JA-Ile. The reduced susceptibility of jinl mutant plants correlates with increased salicylic acid (SA)-dependent signaling. Analysis of jinl plants carrying the sidl mutation, resulting in decreased S A synthesis …

Chromosomes And Expression Mechanisms: Bringing Together The Roles Of Dna, Rna And Proteins, Moshe Yaniv, Sarah C.R. Elgin

Biology Faculty Research

No abstract provided.

The Sacred Emergence Of Nature, Ursula Goodenough, Terrence W. Deacon

Biology Faculty Research

No abstract provided.

Proper Cellular Reorganization During Drosophila Spermatid Individualization Depends On Actin Structures Composed Of Two Domains, Bundles And Meshwork, That Are Differentially Regulated And Have Different Functions, Tatsuhiko Noguchi, Marta Lenartowska, Aaron D. Rogat, Deborah J. Frank, Kathryn G. Miller

Biology Faculty Research

During spermatid individualization in Drosophila, actin structures (cones) mediate cellular remodeling that separates the syncytial spermatids into individual cells. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. We show here that the two domains form separately in time, are regulated by different sets of actin-associated proteins, can be formed independently, and have different roles. Newly forming cones were composed only of bundles, whereas the meshwork formed later, coincident with the onset of cone movement. Polarized distributions of myosin VI, Arp2/3 complex, and the actin-bundling proteins, singed (fascin) and quail …

The Emergence Of Sex, Ursula Goodenough

Biology Faculty Research

Biological traits, the foci of natural selection, are by definition emergent from the genes, proteins, and other “nothing-buts” that constitute them. Moreover, and with the exception of recently emergent “spandrels,” each can be accorded a teleological dimension—each is “for” some purpose conducive to an organism's continuation. Sex, which is “for” the generation of recombinant genomes, may be one of the most ancient and ubiquitous traits in biology. In the course of its evolution, many additional traits, such as gender and nurture, have emerged. Patterns of sexual exchange are the basis for patterns of biological evolution and are central to the …

Drosophila Piwi Associates With Chromatin And Interacts Directly With Hp1a, Brent Brower-Toland, Seth Findley, Ling Jiang, Li Liu, Hang Yin, Monica Dus, Pei Zhou, Sarah C.R. Elgin, Haifan Lin

Biology Faculty Research

The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, we sought a direct link between the RNA silencing system and heterochromatin components. Here we show that PIWI, an ARGONAUTE/PIWI protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic …

Transcription And Rna Interference In The Formation Of Heterochromatin, Shiv Grewal, Sarah C.R. Elgin

Biology Faculty Research

Transcription in heterochromatin seems to be an oxymoron--surely the 'silenced' form of chromatin should not be transcribed. But there have been frequent reports of low-level transcription in heterochromatic regions, and several hundred genes are found in these regions in Drosophila. Most strikingly, recent investigations implicate RNA interference mechanisms in targeting and maintaining heterochromatin, and these mechanisms are inherently dependent on transcription. Silencing of chromatin might involve trans-acting sources of the crucial small RNAs that carry out RNA interference, but in some cases, transcription of the region to be silenced seems to be required--an apparent contradiction.

The Contradictory Definitions Of Heterochromatin: Transcription And Silencing, Kathryn Huisinga, Brent Brower-Toland, Sarah C.R. Elgin

Biology Faculty Research

Eukaryotic genomes are packaged in two general varieties of chromatin: gene-rich euchromatin and gene-poor heterochromatin. Each type of chromatin has been defined by the presence of distinct chromosomal proteins and posttranslational histone modifications. This review addresses recent findings that appear to blur the definitions of euchromatin and heterochromatin by pointing to the presence of typically heterochromatic modifications (including H3K9me) in euchromatin and typically euchromatic enzymes (including RNA polymerases) in heterochromatin. We discuss the implications of these new findings for the current definition of heterochromatin.

Establishment Of Polarity During Organization Of The Acentrosomal Plant Cortical Microtubule Array, Ram Dixit, Eric Chang, Richard Cyr

Biology Faculty Research

The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a …

Comparison Of Dot Chromosome Sequences From D. Melanogaster And D. Virilis Reveals An Enrichment Of Dna Transposon Sequences In Heterochromatic Domains, Elizabeth Slawson, Christopher Shaffer, Colin Malone, Wilson Leung, Elmer Kellmann, Rachel Shevchek, Carolyn Craig, Seth Bloom, James Bogenpohl 2nd, James Dee, Emiko Morimoto, Jenny Myoung, Andrew Nett, Fatih Ozsolak, Mindy Tittiger, Andrea Zeug, Mary-Lou Pardue, Jeremy Buhler, Elaine Mardis, Sarah C.R. Elgin

Biology Faculty Research

Background: Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago. Results: Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of …

The Dot Chromosome Of Drosophila: Insights Into Chromatin States And Their Change Over Evolutionary Time, Nicole Riddle, Sarah C.R. Elgin

Biology Faculty Research

Historically, chromatin has been subdivided into heterochromatin, transcriptionally inactive regions that remain densely packaged throughout the cell cycle, and euchromatin, transcriptionally active regions that take on a diffuse appearance as the cell enters interphase. The banded portion of the small fourth chromosome (dot chromosome) of Drosophila melanogaster is unusual in exhibiting many characteristics of heterochromatic domains, and at the same time maintaining a gene density typical of euchromatin. Similar to genes embedded in pericentric heterochromatin, many of the dot chromosome genes have adapted to a heterochromatic environment. Little is known about the regulation of these genes and less about their …

Reductionism And Holism, Chance And Selection, Mechanism And Mind, Ursula Goodenough

Biology Faculty Research

Despite its rich and deepening panoply of empirical support, evolutionary theory continues to generate widespread concern. Some of this concern can be attributed to misunderstandings of the original concept, some to unfamiliarity with its current trajectories, and some to strongly held fears that it strips the human of cherished attributes. In this essay I seek to deconstruct such misunderstandings, lift up current concepts of what evolution entails, and address some of the existential issues it generates.

Biology Department Publications: 2004, Sam Lindgren

Biology Department Bibliographies

This document is a noncomprehensive list of publications made by graduate students, post-doctoral researchers, and faculty members in the Biology Department in 2004. It indexes those publications available through major databases, and serves as an archival record of the Department’s robust scholarly contributions.

The Cortical Microtubule Array: From Dynamics To Organization, Ram Dixit, Richard Cyr

Biology Faculty Research

Cortical microtubules (CMTs) are essential for normal plant morphogenesis because they affect the axes of cell elongation and predict the placement of cell division planes. The function of the CMTs is intimately linked to their organizational state, which is subject to spatial and temporal modifications by developmental and environmental cues. CMT assembly dynamics govern when, where, and how microtubules appear in a cell's cortex, and the regulation of these properties affects their organization. However, the principles that link microtubule assembly dynamics to cortical array organization and reconfiguration are not well understood. This essay focuses on recent advances in the understanding …

Encounters Between Dynamic Cortical Microtubules Promote Ordering Of The Cortical Array Through Angle-Dependent Modifications Of Microtubule Behavior, Ram Dixit, Richard Cyr

Biology Faculty Research

Ordered cortical microtubule arrays are essential for normal plant morphogenesis, but how these arrays form is unclear. The dynamics of individual cortical microtubules are stochastic and cannot fully account for the observed order; however, using tobacco (Nicotiana tabacum) cells expressing either the MBD-DsRed (microtubule binding domain of the mammalian MAP4 fused to the Discosoma sp red fluorescent protein) or YFP-TUA6 (yellow fluorescent protein fused to the Arabidopsis alpha-tubulin 6 isoform) microtubule markers, we identified intermicrotubule interactions that modify their stochastic behaviors. The intermicrotubule interactions occur when the growing plus-ends of cortical microtubules encounter previously existing cortical microtubules. Importantly, the outcome …

Heterochromatic Silencing And Hp1 Localization In Drosophila Are Dependent On The Rnai Machinery, Manika Pal-Bhadra, Boris Leibovitch, Sumit Gandhi, Madhusudana Rao Chikka, Utpal Bhadra, James Birchler, Sarah C.R. Elgin

Biology Faculty Research

Genes normally resident in euchromatic domains are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by position effect variegation (PEV). Loss-of-function mutations resulting in suppression of PEV have identified critical components of heterochromatin, including proteins HP1, HP2, and histone H3 lysine 9 methyltransferase. Here, we demonstrate that this silencing is dependent on the RNA interference machinery, using tandem mini-white arrays and white transgenes in heterochromatin to show loss of silencing as a result of mutations in piwi, aubergine, or spindle-E (homeless), which encode RNAi components. These mutations result in reduction of H3 Lys9 methylation and delocalization of HP1 …

From Biology To Consciousness To Morality, Ursula Goodenough, Terrence W. Deacon

Biology Faculty Research

Social animals are provisioned with prosocial orientations that operate to transcend self-interest. Morality, as used here, describes human versions of such orientations. We explore the evolutionary antecedents of morality in the context of emergentism, giving considerable attention to the biological traits that undergird awareness and our emergent human forms of mind. We suggest that our moral frames of mind emerge from our primate prosocial capacities, transfigured and valenced by our symbolic languages, cultures, and religions.

Portions of this article were given by Deacon in a paper at the forty-ninth annual conference of IRAS, “Is Nature Enough? The Thirst for …

Biology Department Publications: 2003, Sam Lindgren

Biology Department Bibliographies

This document is a noncomprehensive list of publications made by graduate students, post-doctoral researchers, and faculty members in the Biology Department in 2003. It indexes those publications available through major databases, and serves as an archival record of the Department’s robust scholarly contributions.

The Capacity To Form H-Dna Cannot Substitute For Gaga Factor Binding To A (Ct)N*(Ga)N Regulatory Site, Quinn Lu, John Teare, Howard Granok, Marci Swede, Jenny Xu, Sarah C.R. Elgin

Biology Faculty Research

Previous studies of the Drosophila melanogaster hsp26 gene promoter have demonstrated the importance of a homopurine*homopyrimidine segment [primarily (CT)n*(GA)n] for chromatin structure formation and gene activation. (CT)n regions are known to bind GAGA factor, a dominant enhancer of PEV thought to play a role in generating an accessible chromatin structure. The (CT)n region can also form an H-DNA structure in vitro under acidic pH and negative supercoiling; a detailed map of that structure is reported here. To test whether the (CT)n sequence can function through H-DNA in vivo, we have analyzed a series of hsp26-lacZ transgenes with altered sequences in …

Religious Naturalism And Naturalizing Morality, Ursula Goodenough

Biology Faculty Research

I first offer some reflections on the term religious naturalism. I then outline how moral thought might be configured in the context of religious naturalism. It is proposed that the goal of morality is to generate a flourishing community and that humans negotiate their social interactions using moral capacities that are cultivated in the context of culture. Six such capacities are considered: strategic reciprocity, humaneness, fair–mindedness, courage, reverence, and mindfulness. Moral capacities are contrasted with moral susceptibilities, fueled by self–interest, and brought to the fore in times of stress and humiliation. The essay is in two parts. I first respond …

Biology Department Publications: 2002, Sam Lindgren

Biology Department Bibliographies

This document is a noncomprehensive list of publications made by graduate students, post-doctoral researchers, and faculty members in the Biology Department in 2002. It indexes those publications available through major databases, and serves as an archival record of the Department’s robust scholarly contributions.

Heterochromatin: New Possibilities For The Inheritance Of Structure, Shiv Grewal, Sarah C.R. Elgin

Biology Faculty Research

Significant portions of the eukaryotic genome are heterochromatic, made up largely of repetitious sequences and possessing a distinctive chromatin structure associated with gene silencing. New insights into the form of packaging, the associated histone modifications, and the associated nonhistone chromosomal proteins of heterochromatin have suggested a mechanism for providing an epigenetic mark that allows this distinctive chromatin structure to be maintained following replication and to spread within a given domain.