Zachary T. Goldstein Week 14

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Biological Databases

Part I

  1. What database did you access? (link to the home page of the database)
  2. What is the purpose of the database?
    • The purpose of hPSCreg is to give free access to a global registry of human pluripotent stem cell lines. It allows searching for cell lines and for information available about specific cell lines. An alternative purpose of the database is to register and update new cell lines. hPSCreg aims to collaborate with registries and cell banks worldwide.
    • Link: https://hpscreg.eu/about
  3. What biological information does it contain?
    • The hPSCreg database contains up-to-date, validated information on pluripotent stem cell lines.
    • Link: https://hpscreg.eu/about
  4. What species are covered in the database?
  5. What biological questions can it be used to answer?
    • The hPSCreg database can be used to answer questions about culture conditions for specific cell lines, as well as the availability of cell lines for distribution/purchase. If a researcher wants to culture a certain cell and wants to know if it has been done before/how successful previous trials were, they would use this database. You can search by keyword, specific cell types, or even by diseases. From the hPSCreg database you can also get contact information for cell line distributors.
    • Link: https://hpscreg.eu/cell-line/BCRTi002-A
  6. What type (or types) of database is it (sequence, structure model organism, or specialty [what?]; primary or “meta”; curated electronically, manually [in-house], manually [community])?
    • The hPSCreg database is a specialized database focused on human pluripotent stem cells. It is a meta database that collects (and validates) information from various sources. For example, there is an option to "publish your line" form the homepage. This database is curated online by the scientific community and a committee that validates submitted information before it is added to the database. Information can be updated, edited, and completed through surveys conducted by the Steering Committee and from line providers but only after a validation process and approval from an ethics advisor and Scientific Advisory Board.
    • Link: https://hpscreg.eu/about/documents-and-governance
  7. What individual or organization maintains the database?
    • Maintenance of the registry data is open, fair and scientifically rigorous. It is managed by a "Project management team". Good governance, including the involvement of the Committee of National Representatives, the Ethics Adviser and the Scientific Advisory Board ensures quality adherence. All activities involved in the handling of registry data and information are governed by a Code of Practice and Standard Protocols.
    • Link: https://hpscreg.eu/about/documents-and-governance
  8. What is their funding source(s)?
  9. Is there a license agreement or any restrictions on access to the database?
    • The hPSCreg is a freely accessible global registry with no restrictions, however copies and downloads are only permitted for private, non-commercial use.
    • Link: https://hpscreg.eu/contact/disclaimer
  10. How often is the database updated? When was the last update?
  11. Are there links to other databases?
  12. Can the information be downloaded? And in what file formats?
    • No, there is no clear way to download information from the database which makes sense because it's more of a reference website than an informational website.
    • Link: https://hpscreg.eu/
  13. Evaluate the “user-friendliness” of the database.
    Is the Web site well-organized?
    • Yes the website is very well organized. From the homepage of the database the "browse" and "information" menus are easily accessible and easy to use. The homepage also centrally locates search bars where a researcher can search for specific cell lines or register cell lines of their own. Also included on the homepage is a quick information bit about what hPSCreg is and links to relevant, current news involving the registry. When searching for a cell line the database is quick and efficient, although a lot of information about specific cell lines is lacking in some cases because of community curation.
    Does it have a help section or tutorial?
    • hPSCreg does have a help section but the FAQ link is currently inaccessible. There is information on how to register cell lines, and key search-word examples located just below the search bar on the home page. There is no tutorial option.
    Run a sample query. Do the results make sense?
    • When I searched "United States" in the main search bar 235 cell lines resulted all originating from the United States lined in alphabetical order (as seen by the American flag on the right side of the screen). You can further filter by disease type (ex: Klinefelter) or by uploaded date range (ex: 02/03/03-9/16/16). The results make sense and are easy to understand.

Final RNA Project

Definitions

  1. Trehalose: A sugar of the disaccharide class produced by some fungi, yeasts, and similar organisms. [1]
  2. Nucleolin: A protein found abundantly in the nucleoli of cells, associated with the transcription of ribosomal RNA and the assembly of ribosomes. [2]
  3. Diauxic: the growth in two separate phases due to the preferential use of one carbon source over another; between the phases a temporary lag occurs. [3]
  4. Transient: Lasting only for a short time; impermanent [4]
  5. Glycolysis: The breakdown of glucose by enzymes, releasing energy and pyruvic acid. [5]
  6. Oxidative: Relating to the process or result of oxidizing or being oxidized [6]
  7. Dendrogram: a branching diagram representing a hierarchy of categories based on degree of similarity or number of shared characteristics especially in biological taxonomy [7]
  8. Phosphorylation: Introduce a phosphate group into (a molecule or compound) [8]
  9. Biogenesis: The synthesis of substances by living organisms. [9]
  10. Dimorphic: Occurring in or representing two distinct forms [10]
  11. Proteasome: A complex of proteinases involved in breaking down selected intracellular proteins. [11]
  12. Ubiquitin: A compound found in living cells which plays a role in the degradation of defective and superfluous proteins. It is a single-chain polypeptide. [12]
  13. Glutathione: A compound involved as a coenzyme in oxidation–reduction reactions in cells. It is a tripeptide derived from glutamic acid, cysteine, and glycine. [13]
  14. Glutaredoxin: A family of thioltransferases that contain two active site CYSTEINE residues, which either form a disulfide (oxidized form) or a dithiol (reduced form). [14]
  15. Posttranslational level: Occurring after the translation of a mRNA sequence into the amino-acid sequence it encodes [15]
  16. Molecular Chaperones: A protein required for the proper folding and/or assembly of another protein or protein complex. [16]
  17. Locus Control Region (LCR): A regulatory region first identified in the human beta-globin locus but subsequently found in other loci.[17]
  18. Msn2p/Msn4p- Stress responsive transcriptional activators; activated in stochastic pulses of nuclear localization in response to various stress conditions; binds DNA at stress response elements of responsive genes; relative distribution to nucleus increases upon DNA replication stress [18]
  19. Dimorphic -Having two different distinct forms of individuals within the same species or two different distinct forms of parts within the same organism [19]
  20. Glycogen - A branched polymer of glucose that is mainly produced in liver and muscle cells, and functions as secondary long-term energy storage in animal cells.[20]
  21. Peroxidation - Conversion into a peroxide. [21]
  22. Saccharomyces cerevisiae - Bakers yeast, any of various single-celled fungi that reproduce asexually by budding or division [22]
  23. Escherichia coli - a species of bacterium normally present in intestinal tract of humans and other animals; sometimes pathogenic; can be a threat to food safety [23]
  24. Osmosis/Osmotic -A process by which molecules of a solvent tend to pass through a semipermeable membrane from a less concentrated solution into a more concentrated one. [24]
  25. Chaperone -A protein required for the proper folding and/or assembly of another protein or protein complex. [25]
  26. Kinase -a subclass of the transferases, comprising the enzymes that catalyze the transfer of a high-energy group from a donor (usually ATP) to an acceptor, and named, according to the acceptor, as creatine kinase, fructokinase, etc. [26]
  27. Open Reading Frame (ORF)-
  28. UFD1: Substrate-recruiting cofactor of the Cdc48p-Npl4p-Ufd1p segregase; polyubiquitin binding protein that assists in the dislocation of misfolded [27]
  29. MGA2: ER membrane protein involved in regulation of OLE1 transcription; inactive ER form dimerizes and one subunit is then activated by ubiquitin/proteasome-dependent processing followed by nuclear targeting [28]
  30. NPL4: Substrate-recruiting cofactor of the Cdc48p-Npl4p-Ufd1p segregase; assists Cdc48p in the dislocation of misfolded, polyubiquitinated ERAD substrates that are subsequently delivered to the proteasome for degradation; also involved in the regulated destruction of resident ER membrane proteins [29]

Outline

Introduction
  • Unicellular organisms are programmed to respond to stress
  • Saccharoymces cerevisiae, otherwise known as yeast is studied under different stresses to determine its response on a molecular level
    • Many studies have been done on heat-induced stress
    • Little is known about stress-response to cold
  • Studies have shown that ~10% of the genome changes in response to stress
    • induced or repressed
  • environmental stress response genes (ESR), are involved in many organismal functions
    • regulation of gene is determined by transcription factors Msn2p and Msn4p
  • Adaptation within the cell is determined by different regulatory mechanisms within cells and varies between organisms
  • In S. cerevisiae the genes TIP1, TIR1, TIR2, and NSR1 have been shown to be involved in stress response.
    • S. Cerevisiae wild type and msn2 msn4 cells were analyzed to compare the cold responses to other stressors
Materials and Methods
  • Strains used were the wild-type, BY4743 and BSY25 which was obtained from a cross of two single-mutant strains. W303, a separate wild-type was also used
  • Cultures were inoculated and grown at 30 C on YPD medium.
    • After being harvested during log phase and transferred to new medium, the temperature was decreased 4C per minute.
  • RNA was isolated using the hot-phenol method and purified using the Oligiotex Spin-Column Protocol
  • mRNA was labeled and resulting cDNA was hybridized onto microarrays
  • slides were analyzed within the ScanArray lite scanner apparatus and QuantArray software
    • Normalization was performed for DNA spots to be included in analysis
  • All cultures used in experiment were in the same physiological state
    • Time points used for two independent biological repeats were 0, 2 and 12 hours
    • for three independent biological repeats time points used were 10, 30, and 60 minutes
    • Cy dyes were swapped for reference for each experiment, and a control microarray was done to obtain variability for cultures grown at 30C
      • Only 14 genes within the control demonstrated variability
  • p-value of <0.03 was used for experimental analysis
  • a total of 43 microarray were performed for this study
  • glycogen and trehalose values were determined using a glucose kit
Results
Cold Response of S. cerevisiae
  • S. cerevisiae showed reduced growth rate but a normal growth curve under low temperature conditions
  • The doubling time of cultures with a usual doubling time of ~90 minutes was reduced to 20.7 hours after reducing the temperature from 30 to 10C
  • Cultures reached a stationary phase after ~120 hours
  • A rapid temperature shift from 30 to 10C in S. cerevisiae do show transient changes in gene expression
  • Of the five clusters that were organized using two-dimensional hierarchical clustering, 3 induced genes while 2 repressed genes in response to the cold.
    • One subset of the cold induced genes was particularly induced during the first 2 hours while the other subset was particular induced after 12 and 60 hours.
    • The subsets were labeled as either early cold response (ECR) or late cold response (LCR)
  • A test was performed to determine whether the cold induction treatment was effective and the results showed effective cold induction.
Early Cold Response
  • ECR genes were defined as being reproducibly induced 2-fold or more at one or more of the three early time points examined
  • 130 open reading frames were identified
  • The genes defined are associated with transport, lipid and amino acid metabolism, and transcription, as well as ORFs with unknown functions
  • A set of ECR genes involved in transcription were also defined.
  • UFD1 was significantly induced during the ECR
  • MGA2 and NPL4 showed reproducible increases in transcription abundance during the ECR, but not more than twofold
  • S. cerevisiae cells responded to a temperature downshift as well
    • Levels of expression of some genes rapidly decreased
    • Expression of 32 genes reduced twofold, including genes encoding heat shock proteins
Late Cold Response
Cold Response Compared with Other Environmental Stress Responses
Figure 1
Figure 2

Temperature downshift response yields similar response as early cold shock response. 47% of induced early cold shock genes were induced by the temperature downshift (a). A large amount of down regulated genes in early cold shock were also repressed in the temperature downshift (b).

Figure 3
  • ECR Genes Showed Reciprocal Transcriptional Behavior in Comparison to Other Stress Stimuli. Comparison of ECR genes (CS 2 h) to LCR genes (CS 12 h) and to the responses to other stimuli. Half of repressed ECR genes were induced in heat shock. 40% of induced ECR genes were repressed after 0.5 h of heat shock. 18% of induced ECR genes showed no heat shock response.
  • LCR Genes Showed Similar Transcriptional Responses In All Cases.
  • LCR Involves The ESR and ECR Indicates a Cold-Specific Transcriptional Response. Induced and repressed LCR and ECR genes compared to identified environmental stress response (ESR) genes (Gasch et al., 2000). Induced and repressed LCR genes had a significant overlap of 87 and 111 genes with induced ESR genes. ECR and ESR genes did not have a significant overlap.
Figure 4

Msn2p and msn4p play a major role in late cold shock response and environmental response. Msn2p and msn4p are transcription factors that are required for 99 long cold shock response genes. There are other factors that control late cold shock response. Msn2p and msn4p have little effect in early cold shock response.

Figure 5

Genes Involved in Carbohydrate Metabolism Are Induced at 12 h. •Increase in glycogen and trehalose content observed after 12 h (LCR). Induction of genes in carbohydrate metabolism depend on STREs in the promoters. Mutant strains lacking Msn2p and Msn4p lose induction of these genes during cold treatment.

General Trends in Figure 2-5
  • Majority of repressed ECR genes were also repressed during a temperature downshift from 30 to 10C
  • When the transcription profiles from cultures grown continuously (20 h) at low temperatures (15, 17, or 21°C) were compared with the ECR and LCR profiles, only weak correlations were seen
  • Unexpected correlations observed for OS after 15 min, for MD and XS after 0.5 h, and for DTT after 2 h.
  • Induced ECR genes were repressed, whereas repressed ECR genes were induced
  • ECR and heat shock: Almost half of the repressed ECR genes were induced during heat shock, including HSP genes and genes involved in amino acid and carbohydrate metabolism
  • These observations strongly suggest that the LCR involves the ESR, whereas the ECR indicates a “cold-specific” transcriptional response
  • These results support the comparison of the ECR expression profile to those seen under various stress conditions in indicating a cold-specific response of S. cerevisiae during the early cold response
  • A physiological consequence of the general stress response in S. cerevisiae is the accumulation of the two major reserve carbohydrates, glycogen and trehalose
  • There is no accumulation in response to cold during the first 2 h, but a increase in glycogen and trehalose content was observed after 12 h of cold shock
  • The induction of genes involved in reserve carbohydrate metabolism in response to stress depends on the presence of STREs in the promoters of these genes
Discussion
  • Two distinct responses to cold shock, an early cold shock response (<2 hrs) and a late cold shock response (>12 hrs).
  • Early cold shock response: genes that play role in RNA and fat metabolism
  • Late cold shock response: genes that protect cells
  • Low temperatures slow RNA translation
    • Bacteria cold adapts, ATP-dependent RNA helicases remove mRNA
    • Yeast cold adapts: genes encoding RNA helicases, binding proteins, and processing proteins
    • Mutation in some of these genes leads to cold sensitive phenotypes
  • Cold inducible RNA helicase found in plants too
    • Demonstrates role in cold adaptation
  • Membrane fluidity is another cold adaptation
    • Low temp decreases fluidity
    • Countered by higher prod. of unsaturated fatty acids
      • Involves fatty acid desaturase activity
      • Activity found in bacteria, fish, and yeast
  • LCR gene expression program involves metabolic and stress genes
    • Compensate for reduction in enzyme activity, synthesize protective molecules
  • Trehalose in plants offers cold protection
    • Protects against autolysis, increases freezing tolerance, stabilizes membranes
  • Trehalose and glycogen accumulation observed during LCR
    • Suggested that stress stimulates recycling of both carbohydrates
  • Trehalose observed aiding survival of yeast and E. coli near freezing temperatures
    • May play important role in cold adaptation
    • However, growth rate and viability seemed unaffected by strains w/o trehalose
  • HSP genes found to be cold induced
    • Suggests help is necessary for protein maintenance in cold
  • Global stress-transcription of S. cerevisiae compared w/ gene expression for cold response
    • Data from different labs may have inconsistencies
    • No standard method for inducing stress response
  • Found similarities and differences with different studies
    • Sahara et all (2002) grouping of similar genes found, but observed genes behaving differently
    • May be difference in strain background or experimental design
    • Gasch et al. (2000)
    • Overlap in data found between LCR and ESR genes, indicating LCR activating stress response. ECR response was not consistent
  • Msn2p and msn4p important in glucose and trehalose synthesis in LCR but not the only mechanism
  • S. cerevisiae’s transcriptional cold response comprised of two patterns, early and late
    • Early phase changes membrane fluidity, destabilize RNA
    • Late phase similar to environmental cold response, may be caused by altered physiological state of cell
  • Transcriptional response includes general stress and cold-specific mechanisms.
  • Many other changes likely affect cold survivability of organisms
Figure 6
  • Comparison of Shade and Sahara et al. (2000) yield conflicting and supporting results
  • Comparison of 634 cold-responsive genes
  • Contradiction between the Schade and Sahara et al. (2002) data. Difference found in induction or repression of ribosomal genes.
  • Consistency between the Schade and Sahara et al. (2002) data. Environmental stress response genes being unregulated at times of exposure longer than 2 hours.
  • Are the data publicly available for download? From which web site? Yes [30]

Slides

Schade Journal Club Part I

Acknowledgements

  • I received help on this assignment from Avery Vernon-Moore Courtney Merriam and Jordan T. Detamore in completing definitions and an outline for our journal club paper. We worked in class on definitions and independently on the outline and editing slides.
  • Everything completed on this page is my own work and was not copied from anyone else.

Zachary T. Goldstein 00:48, 28 November 2016 (EST)Zachary T. Goldstein

References




BIOL368/F16

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