62 Matching Annotations
  1. Dec 2022
    1. nuclear pore complex.

      Nuclear pore complex or NPC. There is a size exclusion. <60kDa to pass through unassisted.

    2. The SRP is a G-protein and exchanges its bound GDP for a GTP upon binding to a protein’s signal sequence.

      As soon as this sequence is translated, the SRP binds it. Elongation is temporarily arrested. SRP-ribosome complex binds to the SRP receptor on the ER membrane. Translation now continues into the lumen of the ER. The SPR and its receptor are recycled.

    1. There are a number of ways in which a cell can move from one point in space to another.
      • Axoneme
      • Cell crawling via the remodelling of the actin cytoskeleton.
    1. The modern understanding of the plasma membrane is referred to as the fluid mosaic model. The plasma membrane is composed of a bilayer of phospholipids, with their hydrophobic, fatty acid tails in contact with each other. The landscape of the membrane is studded with proteins, some of which span the membrane. Some of these proteins serve to transport materials into or out of the cell. Carbohydrates are attached to some of the proteins and lipids on the outward-facing surface of the membrane. These form complexes that function to identify the cell to other cells. The fluid nature of the membrane owes itself to the configuration of the fatty acid tails, the presence of cholesterol embedded in the membrane (in animal cells), and the mosaic nature of the proteins and protein-carbohydrate complexes, which are not firmly fixed in place. Plasma membranes enclose the borders of cells, but rather than being a static bag, they are dynamic and constantly in flux.
      • Provides edge to the cell
      • Controls the entry and exit of material.
      • Fluid-mosaic model explains the structure and function of the membrane.
      • High control of intracellular conditions.
    1. In the autumn, histones associated with FLC are acetylated, allowing this repressor of flowering genes to be expressed. During winter, enzymes progressive deacetylate FLC, preventing it from being expressed, and therefore allowing flowering genes to respond to other signals that induce flowering. (Origianl-Deyholos-CC:AN)

      Example of epigenetic change in plants by season.

    1. Crystal violet, CV. In the aqueous solution created, CV exists as its cation CV+. CV+ ions penetrate the cell wall of the bacteria. CV+ binds to negative moieties of the peptidoglycan cell wall. Iodine is added. CV and I- complex and crystallise.

      Decolorization, the variable step, strips off the outer membrane of gram negative, but not gram positive bacteria. As the cell wall in gram negative becomes permeable, CVI diffuses out an colour is lost. <br /> Gram positive retains colour from CVI stain.

      Safarin is a positively charged counterstain. Binds colourless membrane of gram negative.

      Results in pink gram negative and purple gram positive.

    2. The Gram stain procedure is a differential staining procedure that involves multiple steps.

      Gram staining allows for the differentiation of gram positive and gram negative bacteria.

    1. In the early 1900s, the German physician and scientist Paul Ehrlich (1854–1915) set out to discover or synthesize chemical compounds capable of killing infectious microbes without harming the patient.
      • Ehrlich's magic bullet.
      • Salvarsan brought to market in 1910.
      • The systematic selection of compounds, informed by the specific causative agent, still used at present to bring new antibiotics today.
    1. Alexander Fleming: In 1928 Alexander Fleming observed antibiosis against bacteria by a fungus of the genus Penicillium and postulated the effect was mediated by an antibacterial compound, penicillin, and that its antibacterial properties could be exploited for chemotherapy.

      Fleming's serendipitous discovery of penicillin.

    1. certain individuals (van Helmont, Redi, Needham, Spallanzani, and Pasteur) tried to prove or disprove spontaneous generation
    2. The theory of spontaneous generation states that life arose from nonliving matter. It was a long-held belief dating back to Aristotle and the ancient Greeks.
      • In addition, the Egyptians noted that the yearly river Nile mud gave rise to many frogs so they concluded that this mud spontaneously generated frogs.
    1. Mixing does occur between chromosome regions. Not as distinct as first thought.

    2. Subnuclear structure: nuclear bodies * typically spherical<br /> * PML bodies (promyelocytic leukemia protein), involved in transcriptional regulation/cell division * Cleavage bodies * Cajal bodies (CB) * antibody against coilin * Gems * antibody against SMN * CB and gems co-localise with exposure/differentiation to RA. Vary with cell type. * Nuclei of SMA afflicted foetuses lose gems.

    3. Subnuclear structure: Nuclear speckles, not a blanket term. * Also called interchromatin granule clusters * Splicing factor storage (for splicing factors (mostly) not in use) * Proteins may move out of or enter nuclear speckles.

    4. FRAP as a method to test dynamism, movement. Diffusion vs no diffusion of bleached protein. * Mobile fraction * Immobile fraction

    5. 28S, 18S, and 5.8S ribosomal RNA is transcribed (by RNA polymerase I) from hundreds to thousands of tandemly-arranged rDNA genes distributed (in humans) on 10 different chromosomes. The rDNA-containing regions of these 10 chromosomes cluster together in the nucleolus.

      The fibrillar centre is where genes for rRNA are transcribed. The dense fibrillar component where rRNA is processed, chemically modified. The granular component is where protein components are combined with rRNA. Generates preribosomal molecules that are close to being exported to cytoplasm.

    1. Diagram of immunoprecipitation (IP) using either pre-immobilized or free antibodies.

      Immunoprecipitation is a technique for the isolation of protein or a complex (protein-protein interactions) Sample is combined with a specific antibody for the epitope of interest. The antibody-protein complex is removed and analysed.

      1. Molecules from biological sample (lysed) +incubated with antibodies (free or mounted onto support (like agarose bead, magnetic bead))
      2. protein A or G coupled beads added.
      3. Centrifuged
      4. Results in Beads with protein A/G bound to antibody-POI complex.
      5. Well separated in this way, differentially based on sedimentation coefficient.

      Co-immunoprecipitation (can isolate one type of protein in its complex)

      Isolate POI(s) Good with low conc. of POI Protein interactions Unknown proteins Determine if protein is actually being expressed in a given tissue.

      Western blot is carried out to analyse the output.

      Vary salt and detergent levels to preserve or destroy protein interactions.

    1. Export of mRNA and Ribosomes from the Nucleus

      mRNA needs to be assisted across the NPC. Like protein, also classed as facilitated diffusion. * mRNP exporter combines with mRNA with poly A tail, by interacting with FG repeats * mRNA moves through NPC * Dpb5 is an RNA helicase * Straightens the mRNA secondary structure and allows passage, removes proteins on the strand (NXT1, NXF1) * mRNP exporter proteins dissociate from the mRNA. * mRNA is now in cytoplasm

    1. American biologist Lynn Margulis developed endosymbiotic theory, which states that eukaryotes may have been a product of one cell engulfing another, one living within another, and evolving over time until the separate cells were no longer recognizable as such.
      • Margulis hypothesised the popular classical theory for eukaryotic beginnings.
      • Later, as an undergraduate, David Baum proposed the inside-out theory. This is where some eoctye, or archaea, lauched protruding blebs at epibiotic protobacteira. This is how the primitive cell would have harnessed another cell's prokaryotic metabolism for its own benefit, evolving into mitochondria, and in doing so, the inversion of cell membrane into two leaflets via engulfment generates the primitive nucleus -- two layers is still seen.
  2. Nov 2022
    1. Bacterial chromosomes have a single origin, termed ori, whereas eukaryotic organisms can have many not sequence specific.

    2. Can broadly split the process into three parts. Initiation, elongation and termination

    3. DNAPolymerase A

      Primase and DNA polymerase A work in close association.

    4. CDK2

      This is the S-phase CDK.

    5. Assembly of the pre-replicative complex
      • Replication origin licensing.
      • All of this occurs in G1 phase
    6. Orc complex

      Orc1-5 complex.

    7. Cdc6

      Cdc6 is only present in G1 phase.

    8. DNA replication pathway in humans. Orc complex binds the origin of replication.

    1. ChIP

      Workflow 1. Cross-linking 2. Chromatin fragmentation 3. Immunoprecipitation of chromatin 4. DNA recovery and purification 5. Sequencing of DNA

    2. analyze protein interactions

      ChIP-seq is concerned with testing for protein DNA interactions.

    1. NER * Helix distortion * ERCC6 and 8 mutation -- Cockayne's syndrome * XP proteins (XPE or DDB2, XPC, XPA) mutated in xeroderma pigmentosum. * TFIIH, the same helicases as seen in DNA replication.

    1. BER * For non-helix distorting base lesions. * Base is not present, a gap is present in DNA. * Specific DNA glycosylases used for identification. * APEX1 and APEX2 (AP endonucleases): responsible for end processing * An AP site (apurinic/apyrimidinic site). * The exposed 3' OH is available to a replicative polymerase. * Ligation performed by ligase * Short or long patch BER is possible.

    1. In HR, * MRN -- begginings of dsDNA resection. * The PARP1 protein is active on ssDNA. * Free 3' ends made available, crucial for later DNA pol binding. * RPA binds, coats, the ssDNA.<br /> * Rad51 searches for strand for invasion. * Strand invasion carried out by sister chromatid, etc. * D-loop formation.

      May have. * DSBR * SDSA * BIR

    2. NHEJ relies on microhomologies. Doesn't require homologous sequence from another source. * Ku protein instrumental in identification of DSB and recruits DNA-PKcs. * DNa-PKcs autophosphorylates. * DNA ends processed by Artemis. * LIG4 and XCRR4 are needed for strand ligation.

    3. NHEJ and HR can be compared.

    1. TEs are transposable elements.

      Transposons are mobile DNA elements.Can move throughout the genome. Can be catagorised as class 1 (retrotransposons) or class 2 (DNA transposons).

      Class 1 comprises TEs with LTRs, retroposons (LINE), SINEs.

      Class 2 comprises TEs that operate under replicative transposition or non-replicative transposition. Replicative transposition (nick and paste) -- a total of two TEs as an end result, one as part of the donor and one as part of the target sequence. cointegrate.

      Non-replicative transposition (cut and paste) - only one TE generated, in the target.

      Examples of DNA-only transposon:

    1. The initial sample consisted of 1 band. F = 0. 1st generation = The sample overall less dense, still one band. Intermediate density. dsDNA made of one strand heavy and one light. After 2 generations, there was a band for intermediate density and for strands of just light, N-14, dsDNA.

    2. Three methods of replication were initially hypothesized: * Conservative * Semi-conservative * Dispersive

      Semi-conservative method was eventually determined to be correct based on empirical evidence from Messelson & Stahl, in 1958.

    3. Parental strands consist of N-15 isotopes. Replicated daughter strands consist of N-14 isotopes. The CsCl ultracentrifugation process creates density gradient. This allows DNA fragments of different densities to migrate and form a band at the point at which their buoyant density equals that of the salt.

      1. E.coli grown in an heavy nitrogen, N-15, enriched medium
      2. Then transferred to N-14 based media to reproduce
      3. As several points in the experiment, cells were lysed and underwent ultracentrifugation through a CsCl concentration gradient
    1. The several panels show what happens in each cycle. Each cycle consists of a denaturation step at a temperature higher than the melting temperature of the duplex DNA (e.g. 95 oC ), then an annealing step at a temperature below the melting temperature for the primer-template (e.g. 55 oC), followed by extension of the primer by DNA polymerase using dNTPs provided in the reaction. This is done at the temperature optimum for the DNA polymerase (e.g. 70 oC for a thermostable polymerase). Thermocylers are commercially available for carrying out many cycles quickly and reliably
      • Denature
      • Annealing primer
      • Synthesize new DNA with polymerase
      • High speed
      • Extreme sensitivity
  3. Apr 2022
    1. In DNA and RNA, the phosphodiester bond is the linkage between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another, deoxyribose in DNA and ribose in RNA.

      3'-5' phosphodiester linkage

    1. carbon #1, also called the anomeric carbon in carbohydrate terminology
    2. carbon #1, also called the anomeric carbon in carbohydrate terminology

      Carbohydrate terminology

    1. The Strecker synthesis is a two-stage procedure used to synthesize alpha amino acids from aldehydes.

      Synthetic route

    1. The purines have a double ring structure with a six-membered ring fused to a five-membered ring. Pyrimidines are smaller with a single six-membered ring structure. The carbon atoms of the five-carbon sugar are numbered 1', 2', 3', 4', and 5' (1' is read as “one prime”).
      • Purines have a 2-ring heterocycle.
      • Pyrimidines only have one.

      When there is a nucleobase present, the anomeric position on the sugar is labeled 1'

    2. Naming Nucleobases, Nucleosides and Nucleotides

      Nucleosides have a nitrogenous base and a five-carbon carbohydrate group, usually a ribose molecule. Nucleotides are a nucleoside with one or more phosphate groups attached.

    1. Ub-clipping

      Middle down MS technique

    2. branchedubiquitin chains are in fact abundant in mammaliancells and regulate important pathways

      Branched chains contain at least one ubiquitin subunit that is simultaneously modified on multiple acceptor sites, Michael E. French, Chad F. Koehler & Tony Hunter, 2021

    3. K48/K63-branched ubiquitin chains enhance TRAF6-mediated NF-κB signalling by inhibiting K63 chain disassembly.

      NF-kappaB, "nuclear factor kappa-light-chain-enhancer of activated B cells"

    4. ubiquitin biology

      indicated in pathologies such as * cancer * immune defects * neurodegeneration