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This week's reading is Three Habits of Highly Effective Signaling Pathways
Reading Guide Questions
Signaling flips a switch
1. What are the three habits of signaling pathways?
2. What is an SPRE and what is it's significance in signaling pathways?
3. What might be the purpose of experimentally activating a signaling pathway?
4. Define activator insufficiency and zones of competence
5. How do the authors explain the finding that SPREs are insufficient in vitro, but sufficient in cultured cells?
6. What is a signal-independent activator?
7. What is the purpose of co-activators?
8. How do developing organisms solve the problem of leaky expression?
9. Compare two types of default repression and how signaling releases the repression
10. What role does chromatin modification play in develpmental signal transduction cascades?
Class Discussion Questions
Signaling flips a switch
1. Chose one habit to illustrate with a drawing.
2. Explain how Notch, Wnt and Hh accomplish transcriptional switching.
3. What do the authors mean when they say that signaling pathways exhibit "selective transcriptional responsiveness of target genes to pathway activity."?
4. What ways can cells limit the activation of a target gene by an activated signaling pathway?
5. What is a potential drawback to cooperative activation?
6. Illustrate the different types of default repression
7. Illustrate one example of default repression
8. Explain Figure 5: how is it a summary of the output of signaling pathway habits?
9. Explain how Koide et al tested whether default repression is separate from activator insufficiency.
Progressive external ophthalmoplegia
Progressive external ophthalmoplegia is a condition characterized by weakness of the eye muscles. The condition typically appears in adults between ages 18 and 40 and slowly worsens over time. The first sign of progressive external ophthalmoplegia is typically drooping eyelids (ptosis ), which can affect one or both eyelids. As ptosis worsens, affected individuals may use the forehead muscles to try to lift the eyelids, or they may lift up their chin in order to see. Another characteristic feature of progressive external ophthalmoplegia is weakness or paralysis of the muscles that move the eye (ophthalmoplegia). Affected individuals have to turn their head to see in different directions, especially as the ophthalmoplegia worsens. People with progressive external ophthalmoplegia may also have general weakness of the muscles used for movement (myopathy), particularly those in the neck, arms, or legs. The weakness may be especially noticeable during exercise (exercise intolerance). Muscle weakness may also cause difficulty swallowing (dysphagia).
When the muscle cells of affected individuals are stained and viewed under a microscope, these cells usually appear abnormal. These abnormal muscle cells contain an excess of cell structures called mitochondria and are known as ragged-red fibers.
Although muscle weakness is the primary symptom of progressive external ophthalmoplegia, this condition can be accompanied by other signs and symptoms. In these instances, the condition is referred to as progressive external ophthalmoplegia plus (PEO+). Additional signs and symptoms can include hearing loss caused by nerve damage in the inner ear (sensorineural hearing loss), weakness and loss of sensation in the limbs due to nerve damage (neuropathy), impaired muscle coordination (ataxia), a pattern of movement abnormalities known as parkinsonism, and depression.
Progressive external ophthalmoplegia is part of a spectrum of disorders with overlapping signs and symptoms. Similar disorders include ataxia neuropathy spectrum and Kearns-Sayre syndrome. Like progressive external ophthalmoplegia, the other conditions in this spectrum can involve weakness of the eye muscles. However, these conditions have many additional features not shared by most people with progressive external ophthalmoplegia.
Sick sinus syndrome can result from genetic or environmental factors. In many cases, the cause of the condition is unknown.
Genetic changes are an uncommon cause of sick sinus syndrome. Mutations in two genes, SCN5A and HCN4, have been found to cause the condition in a small number of families. These genes provide instructions for making proteins called ion channels that transport positively charged atoms (ions) into cardiac cells, including cells that make up the SA node . The flow of these ions is essential for creating the electrical impulses that start each heartbeat and coordinate contraction of the cardiac muscle . Mutations in these genes reduce the flow of ions, which alters the SA node's ability to create and spread electrical signals. These changes lead to abnormal heartbeats and the other symptoms of sick sinus syndrome.
A particular variation in another gene, MYH6, appears to increase the risk of developing sick sinus syndrome. The protein produced from the MYH6 gene forms part of a larger protein called myosin, which generates the mechanical force needed for cardiac muscle to contract. Researchers believe that the MYH6 gene variation changes the structure of myosin, which can affect cardiac muscle contraction and increase the likelihood of developing an abnormal heartbeat.
More commonly, sick sinus syndrome is caused by other factors that alter the structure or function of the SA node. These include a variety of heart conditions, other disorders such as muscular dystrophy, abnormal inflammation, or a shortage of oxygen (hypoxia). Certain medications, such as drugs given to treat abnormal heart rhythms or high blood pressure, can also disrupt SA node function. One of the most common causes of sick sinus syndrome in children is trauma to the SA node, such as damage that occurs during heart surgery.
In older adults, sick sinus syndrome is often associated with age-related changes in the heart. Over time, the SA node may harden and develop scar-like damage (fibrosis) that prevents it from working properly.
Learn more about the genes associated with Sick sinus syndrome
The long-term outlook ( prognosis ) for people with postural orthostatic tachycardia syndrome (POTS) is generally good, but may be poor in some cases.    While many people have fairly mild symptoms and can continue with regular daily activities, others may be severely affected with limited abilities.  Some people with POTS report significantly improved symptoms within a year,  while others don't improve with treatment and may worsen over time. 
With proper lifestyle changes (for example, exercise and diet) and medical treatments, many affected people see an improvement in symptoms and quality of life. In some cases, people with POTS may even become symptom-free over time.  
Taken together, these linked laboratories offer a high-risk/high-reward research agenda for the training of young scientists. In addition to imparting multiple bioinformatics and molecular biology techniques, the activities empirically demonstrate a major finding of developmental biology and comparative genomics namely, that the inner molecular pathways of organisms are not as diverse as the organisms outwardly appear. The choice of signaling pathway and species is flexible, offering endless variations on this core biological theme. The results of the project permanently increase the global store of comparative data on genetic sequence and expression. These common fruits can then be productively picked for future studies on organisms' genetic, epigenetic, and evolutionary relationships.
4.E: Genetic Toolkit Exercises - Biology
This toolkit contains links to tools, resources, and information about Environmental Sensitivity Index (ESI) maps and data.
ESI maps and data are available in a variety of formats and can be downloaded for free. The maps can be viewed and printed, and the data can be used in GIS technology. In addition to the links in this section, you may also want to review the ESI Documents section to obtain ESI products.
Download ESI Maps and GIS Data: Download maps and data in a number of formats.
Availability: Find out what regions have been mapped using the ESI method and download metadata, indexes, and fact sheets for each ESI atlas.
These resources describe how ESI maps are developed and how to use them.
ESI Maps: Read an overview of ESI maps.
Anatomy of ESI Maps: Learn about the basic elements of ESI maps, including examples of the front and back of a map and an overview of how the maps are created.
Shoreline Rankings: Under the ESI method, shorelines are ranked according to their sensitivity to oil, the natural persistence of oil, and the expected ease of cleanup after an oil spill. In this series of pages, get an overview of the shoreline sensitivity rankings, a table of shoreline ranks, and an example of a shoreline ranked 10A.
Biological Resources: On ESI maps, the biological resources that are mapped include oil-sensitive animals and their habitats, and habitats that are themselves sensitive to spilled oil (such as coral reefs). In this page series, get an overview of biological resources and a table of symbols and patterns used to designate them.
Human-Use Resources: On ESI maps, the human-use resources that are mapped are resources and places important to humans and sensitive to oiling--such as public beaches and parks, marine sanctuaries, water intakes, and archaeological sites. This series of pages includes an overview of human-use resources and a table of symbols used to designate them.
You can use the ESI tools to assist you in viewing and querying ESI atlases that have been published in geodatabase format. You can download them individually or all together in the ESI Toolbar. We recommend downloading the toolbar, because the tools complement each other.
ESI Toolbar: Overview of the ESI tools, and information on how to download the ESI Toolbar (which includes all of the ESI tools).
Query by Location Tool: This tool provides a drop-down menu of the available biology and human-use layers on the ESI map. After you select a layer, you can view the species or the types and names of the socioeconomic and management features present in a particular area. You may also set the fields in the biology and socioeconomic tables that you would like to view.
Query Biology by Attribute Tool: This tool allows you to query a biology layer by attribute (such as species, state and federal status, and monthly presence and/or breeding status). With this tool, you can locate species of special interest in a particular area, such as where a spill trajectory indicates that there may be heavy oiling.
Seasonal Summary Tool: This tool summarizes a region of interest and allows you to generate a text report, an 8.5 x 11 inch map layout, and a new geodatabase that contains the requested subset of feature layers and tables.
Report Generator Tool: This tool allows you to generate a Resource at Risk report, by exporting records you have selected in your biology table to a tab-delimited text file.
Metadata Viewer Tool: This tool launches your PDF viewer software and opens the metadata file associated with your ESI map. The metadata file is provided in the atlas directory transferred from the ESI CD/DVD.
Science Choreography: A Movement-Based Approach to Biology Teaching
We would like to bring to the attention of readers a novel participatory movementsed technique we have been using to teach science and to encourage students to think creatively about science: science choreography. The project evolved as part of a Howard Hughes Medical Institute𠄿unded multiyear collaboration between a team of scientist-educators at Wesleyan University and other institutions and dancer-choreographers from the Liz Lerman Dance Exchange. When the sequencing of the human genome was announced to the public, choreographer and MacArthur Fellow Liz Lerman was one of many who asked what this might mean for our future and the future of our children. To help answer these questions, she decided to make a multimedia piece, Ferocious Beauty: Genome, in collaboration with scientists at Wesleyan University and across the country ( Science Choreography, 2011 ). After the work premiered, it became evident to us that we could use video performance clips from Ferocious Beauty: Genome as a “second textbook”𠅊 launching point and source of enrichment for learning and thinking about concepts in biology. We also came to realize that a number of the tools used in dance making by the Dance Exchange could be easily adapted for use in the classroom, either alone or in combination with video clips from Ferocious Beauty: Genome.
We were motivated by a variety of challenges faced by science teachers at all levels, including the perception by some that our subjects are difficult to teach and hard to learn, as well as threatening or uninteresting to many. Our immediate goal was to make science more accessible to a broad base of students. One target student is the kinesthetic learner ( Gardner, 1983 Snyder 2000 ). The value of a movement-based approach in reaching a diverse student body is underscored by the observation that embodied learning is particularly effective at engaging at-risk teens and ethnic minorities ( Park, 1997 Tanner and Allen, 2004 ).
Our work together has generated a website for science educators from middle school through college ( Science Choreography, 2011 ). It provides a rationale for the approach descriptions of a toolbox of teaching and learning exercises, including video demonstrations and examples of content-based modules we have developed on a number of topics. Imagine students who have recently begun to explore concepts of genetics—phenotypes, genes, genetic crosses—meeting with Gregor Mendel, the father of genetics, to discuss his experiences as a founding scientist in biology. What was he thinking? What led him to design his classic experiments? How is modern biology dependent on Mendel's pioneering work? What is a gene? These are some core questions that students and teachers can raise in the Genes and Mendel module, which includes a clip from Ferocious Beauty: Genome that features a dancer in the character of Mendel. In another module, we focus on bringing ethical considerations into biology teaching, a challenge for which science teachers may feel they do not have the necessary expertise. In the Ethics and Genetic Testing module, we use two tools: Ask a Question in small groups or Walk and Talk in larger settings, to transition from a lecture-style, PowerPoint-based presentation on the “why, what, when, and how?” of genetic testing to thinking and talking personally about related ethical issues, with questions such as “Would you want to get a genetic test for a disease?,” What if you were predisposed to it?,” “Who should know the results?” We also view a relevant segment of Ferocious Beauty: Genome that features a performer with osteogenesis imperfecta who dances in a wheelchair and on crutches. In the DNA Helix module, the aim is for students to learn DNA properties from one another and engage in model building, but rather than using the machined metal pieces Watson and Crick used, they use their own bodies ( Figure 1 ). By doing this in stages, the students can start to understand more viscerally what the structures are, and how some models are more robust than others. The embodying tools are designed so they can be used independently of the specific modules, for example, as a kinesthetic mnemonic to help students remember a pathway or as a means for an instructor to find out what students know about a specific topic. Getting the students up on their feet in the midst of a conventional sedentary classroom setting is restorative and invigorating.
ASSESSMENTS OF LEARNING OUTCOMES
This bioinformatics component definitely increased student awareness of bioinformatics as an emerging field. Results of our survey in Spring 2010 indicated that none of the entering freshmen had heard of bioinformatics and that they first became aware of the field and its essential role in modern biology through this course. We were also under the impression that students in general had shown increased enthusiasm for learning biology, which was reflected from their active participation in the class and their expression of interests in taking more courses in this area, such as Introduction to Biocomputing and Molecular Evolution. Student performance was evaluated mainly through examinations, laboratory reports, and a class project. For example, students were given a set of questions for each lab which was designed to assess the level of their understanding of the concepts and skills they learned. All of the students were able to complete the assignments with help from the instructor. For the projects, each student was asked to focus on a particular genetic disorder based on his or her own interests. The project assessed each student's ability to independently search databases to retrieve genetic sequences and other information associated with the disorder and analyze the genetic data using bioinformatics tools and software. All of the students were required to write a paper on their projects to present their findings. Through the project, students had investigated a wide range of genetic disorders using bioinformatics tools, including sickle cell anemia, Huntington disease, albinism, breast cancer, and many others. Seventy to eighty percent of the students were able to complete their projects satisfactorily.
Each of the three modules is represented by the QPCR results in Table I. The Ct represents the number of cycles of QPCR required for each sample to get to a geometric phase of amplification, also called the threshold. A low Ct represents more DNA amplification versus a higher Ct in the same experiment. The genotype examined is a VNTR thus, there will be a longer stretch of DNA and a lower Ct (more fluorescence) for the longer 5/5 genotype than there would for the 4/4 genotype as long as the same initial amount of template DNA is used. As indicated in Table I, there are clear differences among the three possible genotypes in the QPCR module (as indicated by the difference between the Cts). Some of the results of the QPCR module were confirmed with the traditional PCR gel electrophoresis module (Fig. 2, samples 1, 3, and 5). The gel shows each of the three possible genotypes (both homozygotes and the heterozygote) in the appropriately labeled lane (Fig. 2). It is interesting to note that the 4/5 heterozygote did not run neatly on the agarose gel, although the QPCR results confirm what appears to be a heterozygote on the gel. This type of ambiguous electrophoresis result can occur quite often, and it is very useful to have two different ways to confirm a particular data point.
|Sample||5/5 Ct||4/5 Ct||4/4 Ct||Per3 cDNA Ct||Bactin cDNA Ct||ΔCt|
- An undergraduate biochemistry laboratory section at Lawrence University performed the modules described above with one replicate each. The Ct for the DNA of each volunteer is shown for the three possible genotypes 4/4, 4/5, and 5/5. The Per3 Ct refers to the gene of interest and the Bactin Ct is for the housekeeping gene. The ΔCt is calculated as the Ct of the gene of interest minus the Ct of the housekeeping gene
The second module examines the Per3 mRNA expression level of each individual. The expression of a gene is influenced by the individual's genetics makeup and environmental factors. When determining the expression level, the quality of the total RNA isolation needs to be controlled for by measuring the expression level of a housekeeping gene whose expression should remain constant. The Ct of the housekeeping gene is subtracted from that of the gene of interest to give a ΔCt. For this experiment, the data (Table I) shows the variability between individuals. It may be interesting for students to attempt to correlate mRNA expression levels with a variety of environmental factors including the quality of the individual's sleep (see supplemental sleep survey).
The third module reflects another influence of the environment upon gene expression, methylation. Methylation of a gene reduces mRNA expression of that gene. The Per1 gene was used for this module because there is generally a larger difference in methylation patterns of the Per1 gene (particularly age related) than those of the Per3 gene and both are in the same gene family. The sample tested with primers that were designed to anneal to DNA that had not undergone bisulfite conversion had a lower Ct, 24.44, than the converted sample Ct of 33.61. This indicates that less amplification of the converted sample (where methylated cytosines are converted to uridines) was achieved because the primers could not anneal precisely to the template DNA, and therefore, there was relatively little methylation. Thus, the use of the epigenetic module affords an excellent opportunity to discuss the chemistry and kinetics of primer hybridization in addition to a discussion of methylation as a means of transcriptional control.
Cellular and Molecular Toxicology
G. Leikauf , K.S. Ramos , in Comprehensive Toxicology , 2010
Functional genomics have been validated and is currently gaining wide acceptance in toxicology. This chapter focuses on transcriptomics, but similar approaches are being applied in the areas of proteomics and metabolomics. Such investigations now are improving our knowledge of the underlying biology and the regulatory networks that integrate the signaling cascades involved in toxicity. These methods provide novel insights into the mechanism of action, especially when pared with classical validated toxicological methods. Toxicogenomics will further advance the introduction of mechanistic insight into risk assessment and fulfill the promise of more accurate determination of class-related biological effects or predictive toxicity. Although, some have proposed that functional genomics will replace existing approaches in toxicology ( Collins et al. 2008 ), it remains critical to integrate data from multiple sources to produce a comprehensive understanding of the molecular basis of adverse responses ( National Research Council 2007 ). For this reason, improved algorithms must be developed that combine and interpret data of multiple types (e.g., gene-expression, proteomic, and metabolomic data). Integration of data from different technologies will lead to synergistic interpretations beyond what can be resolved when data are analyzed in isolation. Examples include the interplay between transcriptional analysis of protein factors and gene-expression changes and between levels of metabolizing enzymes and the production or elimination of metabolites.
The data-rich nature of toxicogenomic technologies, coupled to challenges of data interpretation, make the application of toxicogenomics in risk assessment inherently complex. Nonetheless, advances in data visualization methods and integrated assessments across technologies are progressing rapidly. In addition, a pressing need in genomics is for more accurate identification of orthologous genes or proteins across species. This effort will improve our understanding of conservation of biological responses to toxic injury and facilitate the use of surrogate species that predict the responses in humans. Although species differences in proteomes may be large, many responses to chemical and physical stressors are evolutionarily conserved, and limitations posed by cross-species extrapolation can be mitigated by focusing analyses on processes conserved across species. However, most protein networks of rats, mice, and humans remain uncharacterized, and divergence can be a major factor in species differences in sensitivity or response.
Pathogenesis is typically defined by a linear sequence of events. In contrast, system biology predicts nonlinear cellular states of compensatory feedback loops that depict the true complexity of an organism. The development of a knowledge base to accurately reflect network-level molecular expression presents large challenges to data management, integration, and computational modeling. As these fields advance, the development of approaches that combined model systems and interactome analyses could unravel the complexity inherent to biological systems. The application of functional genomics to the study of mechanism of action has advanced our understanding of the biology that underlies the deleterious actions of chemical and pharmaceutical agents on living systems, the regulatory networks that integrate the signaling cascades involved in toxicity, and the pathogenesis of environmental or drug-induced disease. Indeed, mechanistic toxicology investigations have proven useful in risk assessment, drug development, environmental exposure assessment, and understanding of human and animal variability in response to drugs and chemicals. Progress to date has been limited by the scarcity of comprehensive time- and dose-related investigations and the lack of studies using exposure paradigms that reproduce the human condition with fidelity. Nonetheless, this area of science is growing rapidly and the application to better understanding the mechanism of action of drugs and chemicals will be widespread.