February 15-19 Notes
February 15-19 Notes CELL 2050
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Joseph Merritt Ramsey
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This 18 page Class Notes was uploaded by Joseph Merritt Ramsey on Monday March 7, 2016. The Class Notes belongs to CELL 2050 at Tulane University taught by Dr. Meenakshi Vijayaraghavan in Winter 2016. Since its upload, it has received 30 views. For similar materials see Genetics in Science at Tulane University.
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Date Created: 03/07/16
February 17, 2016 Chapter 10: Chromosome Organization and Molecular Structure Chromosome Overview o Function 1) Genetic Storage 2) Genetic Expression 3) rRNA Assembly 4) Compacting and Sorting o Characteristics Bacteria Eukaryotes Viruses o “Bad news wrapped up in a protein” o Overview Genetic material determines the shape and size of a virus Viruses need a host cell in order to infect This infection id dependent on the Host Range Host Range is in essence the ability for the host to be infected Primary and Secondary Hosts allow for viral emergence (passing it on) o Structural Set Up Simple set up has the same protein subunits surrounding the genome Complex structures: 1. Have any sort of genetic material (RNA or DNA, single or double) 2. Usually have multiple protein types on the coat (Even Phages, they have several types of proteins) Membrane viruses They obtain the membrane as they enter into the cell Spike Proteins allow for entrance (by binding to host) o H (Hemaglutinin) – the virus can gain entry o N (Neuraminidase) – the virus can exit Our antibodies recognize the Spike Proteins, but they can rearrange to create new strains o The different combinations of spikes creates new virus strains unfamiliar to the body Membrane Creation o 1. Internal – Cell Protection, replication includes membrane o 2. External – Macrophage Protection, as they exit they obtain a membrane o Cycle Types 1. Lytic Very often Bacteriophages As soon as they enter the cell: o I. Replicate Genetic Material o II. Assemble Proteins o III. Aggregate Viral Particles (Either Self or Directed Assembly) o IV. Exit Through Lysing These are known as Virulent Viruses 2. Lysogenic Also known as Temperate virus o A dormant stage appears Process: o I. Insert’s Viral Genome into Host Genome This inserted portion is no known as the Prophage (viral genome inserted into the host) o II. Prophage is Replicated With the Cell o III. Signal Initiated Viral Exiting The viral genome (Prophage) suddenly leaves Moves into assembly phase (transcribed) o IV. Now Assembles Transcribes and assembles proteins and creates viral particles Now is becomes a Virulent Virus, Lytic *So every virus will eventually enter the lytic phase* HIV Example o Follows Lysogenic cycle for a bit in the CD4+ cells – creates Prophage o They then eventually begin to Bud (almost like leaking) from the cell, a process that is not virulent But particles are present in the bloodstream They traverse the whole body and infect new cells o They then move into the TCells where the lytic phase begins – breaking down of TCell, leading to cell count decline o So what is considered infection? When they are capable of making viral particles Vaccines attack the Protein Coat for the most part So we can stop (vaccines): 1. Genetic Material Replication 2. Protein Forming o Genomes Can be Single/Double, RNA/DNA Few 1,000100,000 nucleotides Hold only a small number of genes (normally 520) T2 Even Phages hold the most number of genes and are therefore the most complex (About 200) Various protein options to make up external membrane o Assembly – viral particles have to assemble themselves (Capsid) 1. SelfAssembly Very simple organization Genetic material is coated in single protein, made of identical protein units Tobacco Mosaic Virus o 2100 Copies of the same protein o Single strand of RNA 2. Directed Assembly Generally a more complicated genome (T2 Even) with several types of proteins Process o 1) Assembly of Different Types of Proteins – done by scaffolds that don’t belong to the virus Diagram o 2) Protein Lysis Takes Place – they are broken down, cut (by Proteolytic Enzymes) to fit function Also done by host cell Cut to be assembled into Capsid Bacteria o Chromosome Overview Millions of Nucleotides Naked DNA in the Nucleoid Region Circular, Double Stranded **Only have one chromosome replication point (as opposed to multiple in Eukaryotes) Roughly 100200 bp long for the Origin of Replication Several genes are present with multiple copies Ones involved with Gene Expression especially, which are related to the points below 3 R’s of Purpose o 1. Replication o 2. Recombination o 3. Regulation Compaction o Compaction Originally formed in 110nm, need 1000fold compaction Loop Domain Formation (Step I) Size of the loop is contingent on Genome This is a 10Fold level of compaction Radial Loops occurs in Eukaryotes Super Coiling The next step, occurs within the loop itself DNA is a right handed molecule 10bp/turn is the only stable structure for DNA Left Right Occurs on areas that need to be Occurs on areas that are less expressed – this is the negative coil frequently expressed – the positive coil Adding Pressure/Twisting results Adding pressure/twisting results in fewer turns in more turns (becoming tighter) So the average bp becomes 12.5 in So a fewer bp average is created initial form, which does not fit the (8.3/turn) in the initial coil proper stability So the loop coils to return to So the loop coils to returns to 10bp/turn 10bp/turn Known as a Positive Supercoil (a Known as a Negative Supercoil (a left twist) right twist) Topoisomerase II (Gyrase) Topoisomerase I Induces negative supercoiling Induces positive supercoiling and relaxes positive supercoiling and Relaxes negative supercoiling This is the important one for With minimal activity acts to undo compaction of Euchromatin (no Negatively supercoiled regions more expression) Then they work in conjunction – replication can occur because of loosening by Topoisomerase II (Gyrase), negative is easier to open up and access So both need to be functioning properly to coordinate action Similar comparison to Heterochromatin and Euchromatin Unwinding Process of Positively Supercoiled Regions (tighter) – Topoisomerase II (Gyrase) “cuts and pastes” the DNA 1. DNA loops around A (holds DNA, bottom structure) and then wraps around B (formation of loop, top structure) 2. A makes the cut (lower unit) 3. Loop from B subunit is released and “slides down” through the cut 4. DNA rejoined *Lower Jaw cuts using energy from 2ATP binding in the BSubunit* *1 cut and paste forms 2 Loops* o How Do Antibiotics Work? Not in Extreme Concentrations, Two Mechanisms 1. Quinolones These induce structural organization of DNA So in essence, preventing unwinding They stay compacted and inaccessible 2. Coumarins Act as competitive inhibitors on the BSubunit where ATP binds to induce the Gyrase action So the wrapping around Gyrase can still occur (this is contingent on protein structure) But the binding site for cutting action is unavailable, so A does not cut No Cut = No Loops = No Completion of Replication o Topoisomerase 4 also acts in similar manner as I to help replication Eukaryotes o Chromosome Overview Billions of Base Pairs (bp) Chromatin is the functional part of the chromosome Protein (Histones) + DNA Most are large, but size does not equal complexity Introns are present that give size Repetitive Sequences are present Long and Linear o Important Characteristics of the Chromosome 1. Origins of Replication Eukaryotes have multiple origins These larger number helps increase Replication Efficiency of chromosomes o Because Eukaryotic chromosomes are so much longer Once every 100,000bp an Origin Shows up 2. Centromere Intrinsically involved in sorting o Kinetochore deposition occurs Marked by specific Sequence o Point Sequences occur in simple organisms (125 bp in length) o Regional Sequences occur in complex organisms (made of Tandem Repeats, 914bp, to total up to a large sequence), add up to millions o Centromeres are Heterochromatic Sequences, which often have repeats (CG, more dense) Also has histones o Core histones o H3 is replaced during replication 3. Telomeres February 19, 2016 Chapter 10: Chromosome Organization and Molecular Structure Chromosome Overview Viruses o “Bad news wrapped up in a protein” o Overview o Structural Set Up o Life Cycle Cycle Types 1. Lytic 2. Lysogenic Genomes Assembly Types – viral particles have to assemble themselves 1. SelfAssembling 2. Directed Assembly Bacteria o Chromosome Overview o Compaction o How Do Antibiotics Work? Eukaryotes o Chromosome Overview o Important Characteristics of the Chromosome 1. Origins of Replication 2. Centromere 3. Telomeres – important for stability I. Protection o Nucleases act in the body, targeting DNA splicing (they digest DNA) o They act more readily on peripheral DNA, Exonuclease o Endonucleases are more medial regions o Telomere regions have sequences that throw off Exonuclease recognition II. Repair o They also act to repair Chromosomes III. Aging o Aging is associated with Telomere shortening o They maintain chromosome length in this manner IV. Heterochromatic Region o Overall Structure Length of Chromosome determines how many genes can fit Chromosome 1(longest): 215mbp 4316genes Chromosome 22 (shortest): 50mbp 500600genes Heterochromatic Regions Always in the Centromere and Telomere But also present in particular regions along chromosome o Repetitive Sequencing Sequence Complexity – deals with the number of times a sequence appears along the genome Types of Repetition: I. Unique (41%) o Three Types of Unique 2% are Protein Encoding/Structural 24% are Introns (intervening sequences) 15% are unique and Not Found Within Genes (specific functions, regulation, imprinting control regions, for example) o Only present a few times in the genome o We only have less than 100 of unique repetitive II. Moderately Repetitive o A few hundred copies of these sequences are seen in the genome 1500 genes o 1. Functional A good example is the origin of replication These are seen every 100,000 bp Often genes that are needed in relatively large volume (so they can be expressed a lot) rRNA Histones Origin of Replication All involved with expression o 2. May Not Have Function Transposable Elements are an important type – they may not function TEI – uses reverse Transcriptase, so RNA becoming DNA then inserting, retrovirus TE2 – Transposase, Copied as DNA and inserted as DNA, most Mutations occur in this type They are capable of moving around III. Highly Repetitive o Tens of thousands or even millions of times o Relatively short copies, a few hundred bp (10 400max) o Present every 50006000 nucleotides o Most of the time, don’t have an explicit function simply because of sheer volume o Usually Heterochromatic o Alu 300bp, every 5000 nucleotides Restriction enzyme break site How do we test for repetitive sequences? The process utilizes Cleavage and Density properties of DNA I. Cleavage o H1 bonding site cleavage 600bp length segments o Then the fragments are heated up to break the HBonding in the DNA, making the strand single stranded o Slowly return to normal temperature, allowing the double helix to reform (HBonding) Highly repetitive reform first because they 1) 2) are Smaller Ubiquitously Present (so the probability is increased) Anele Rate (reforming rate) of the more present sequence is higher II. Density o A’s and T’s (associated with Highly Repetitive Sequences) are less dense Tandem Repeats are associated with A and T (Heterochromatic) G’s and C’s are usually associated with Functional Genes o Done through CesiumChloride Density Gradient Add mixture with known CsCl Density Centrifuge to forms density bands o Compaction in Eukaryotes Why do they need to compact? We have a very long, 12 meters long to fit into 2 4micrometer region of the nucleus Process Overview I. Initial 7Fold Compaction (Mardi Gras Beads), the Nucleosome Model o This is the wrapping o 2nm Diameter 11nm Diameter o Histone Proteins (2x Each to Form Octamer) H2A – bound to tail H2B – only part of Globular region H3 H4 o Octamer Globular Domain – basophilic Amino Tail – positively charged amino acids Has Lysin and Arginine 146147bp wrap around each Octamer 1.65 Negative turn o Arginine extends Electrogenic and HBonds towards the Phosphate (–) for binding This allows the wrapping to occur around the octamer This forms the Nucleosomes, making the Bead Structure o Nucleosome Chromosome is a complex that is made of repeating units of Nucleosomes 200bp each H1 is the fifth histone present Linker DNA is easily accessible o Experiment proving Histone Model DNASE1 breaks down DNA So varying concentrations should break fragments to make 200bp fragments He would expose to the nucleus Higher concentration cuts more cleanly So bands form in GE because of varying cleavage locations All bands were multiples of 200 II. Secondary 7Fold Compaction (Beads Clumping Together), Contingent on H1, so the H1 Model o 11nm 30nm (another 7fold compaction, so 49 total right now) o H1 reduces the space between the two nucleosomes o How did we learn about H1? 100mM Na/Cl can undo H1 association Removal restored 30nm fiber o How is 30nm formed? The reduction is done in a random manner, leading to ZigZag Model They are not arranged in a certain order o This 30nm level is common to ALL DNA But after that differentiation of compaction changes III. Radial Loop Formation o This is where the formation of Euchromatin and Heterochromatin occur (lightly stained, darkly stained) o Similar to Loop Domains in Bacteria Compaction Comparison Euchromatin Heterochromatin Lightly stained (less compact) Darker Stain (more compact) Transcriptionally Active Transcriptionally Inactive 30nm fiber forms a 300nm Radial Supercoiling occurs to give a very Loop tight structure o 300 nm Level of Compaction o This changes the overall (*Diameter of The Loop) diameter (of the “thread”) o 30nm Diameter (*Diameter of Simply has a 700nm diameter the “Thread”) fiber, *no level of compaction to Most chromosomes are largely describe euchromatic o Not as wound up to allow access How Does it Maintain the Loop? – The Role of the Nuclear Matrix Compacting chromosomes latch onto the Nuclear Matrix o Outer Matrix – Nuclear Lamina, Intermediate Fibers giving support to Inner Nuclear Membrane o Inner Nuclear Matrix Proteins – Scaffold of proteins of irregular length DNA has specific sequences that bond with the Inner Nuclear Matrix Proteins o They use Linker Proteins to Bind to Matrix Proteins with DNA regions o MARS – Matrix Attachment Regions o SARS – Scaffold Attachment Regions Features of the Loop o 20,000 to 250,000bp are present in each loop o Cannot break the Linker Protein attachment February 15, 2016 Chapter 3: Cell Division (Cont.) Genetic Material Cytogenetics Eukaryotic Chromosomes Mitotic Stages Sexual Reproduction o Gametes o Meiosis preceded by Syngamy (germ cell fusion, fertilization) This: 1. Keeps Consistent Chromosome Number 2. Brings About Variation to Increase Survival Interphase still occurs in Meiosis It has two rounds of division, so a sort of Inter Interphase Exists o Interphase II has G1 and G2, but no S Phase Results in the creation of 4 Genetically Inconsistent Haploid Cells (small allelic differences exist) But overall, the divisions are the same Simply processed by Reduction Division o Meiosis o Spermatogenesis Testes have Spermatagonial Cells, which are selfrenewing cells This is a Diploid Cell (Spermatagonia) These cells divide (through Mitosis) to produce two new cells, one that remains a Spermatagonial Cell and one that becomes the Primary Spermatocyte The Primary Spermatocyte goes through Meiosis to produce Gametes After Meiosis I the Primary Spermatocyte has been split into two Secondary Spermatocytes The final phase (Meiosis II) results in four Spermatids The spermatids go on to differentiate into the Sperm Cells The main goal of Spermatogensis is to produce haploid nuclei What does Sperm Differentiation add? I. Motility o The develop a flagella, helps motility II. Enzyme Capsule o This is known as the Acrosome (enzyme capsule that breaks down egg membrane) o Oogenesis Very important because the cell must initiate and sustain embryonic development Accumulation (this is a much more complex process) for Embryo I. Cytoplasm and Enzymes II. mRNA III. Proteins IV. Organelles Creation Process Special diploid cells in the Ovary are called Oogonia o Mitosis is the first step again o But this isn’t SelfRenewing (constant division) Oogonia develop into Primary Oocytes and an additional Oogonia o This one will go through Meiosis I, but in an Asymmetrical manner o Spindle Apparatus forms to one side, and organelles follow, forming Secondary Oocyte and a Polar Body (which divides again) By month seven, however, the addition Oogonia degenerate and leave the Primary Oocytes Through a long activation process, the Primary Oocytes go on to become Three Polar Bodies and A Functional Egg Activation Process I. Early Development o Through Mitotic Proliferation of early ovary cells, about 7 Million Oogonia form o As development continues, this number declines to roughly 1 Million Functional Oogonia (undergo Meiosis) II. Selection Process (7 Months) o At Seven Months, a selection process occurs to the 1,000,000 that results in 400500 Primary Oocytes III. Diplotene Arrest (Primary Signal, 7 Months, Just Before Birth) o At the same time as selection occurs, Primary Oocytes begin to undergo Meiosis o They are halted at Diplotene, however, in Prophase I o It’s important to note that Prophase has begun, so the spindle apparatus has formed That’s why age creates problems IV. Second Signal (12 Years, Puberty) o Hormonal shifts result in Meiosis progressing through to Metaphase II Occurs in monthly cyclic process o So the first division has occurred, giving rise to a Polar Body V. Final Signal (Sperm Cell) o Sperm cells arrive and attack the membrane of the Egg in order to fuse o This signal induces completion of Meiosis and creation of egg cell and ultimately embryo o Plant Gametogenesis (Double Fertilization) Methodology of Alternation Gametophytes are the Haploid Phase, Sporophyte the Haploid Phase Large visible structures are generally Gametophytes Creation Spores Eggs Anther, Stamen (producer) = Male Stigma, Ovary (Producer) = Female Diploid Parts Diploid Parts I. Microsporocyte (Meiosis) I. Megasporocyte Megaspores (4) Microspores II. Three Megaspores Degenerate II. Microspores Pollen Grain This division occurs without Cytokinesis III. Three Rounds of Mitotic Division and Unequal Cytokinesis III. Pollen Grain is made of Tube Cell ultimately result in Seven Cells (Pollen Tube) and Generative Cell (Embryo Sac) (one is Binucleate) (Active Sperm Cells) Egg, Central Cell (Binucleate), Synergids (2), Antipodals (3) Fertilization I. Stigma (Female Part) o Nectar and lipids present on tip o Lipids and nectar initiate germination, maturation of pollen grain II. Pollen Grain (Male) (Pollen Tube and Sperm Cell Nuclei) o Tube Cell – develops into the pollen tube o Generative Cell – splits into two germ cells (mitosis) to become active sperm (sperm nuclei) III. Newly Created Sperm Cells Fertilize (Endosperm and Embryo, this is the Double Fertilization) o Central Cell (Female) – this Binucleate cells now becomes a large trinucleate, known as the Endosperm o Egg Cell (Female) – fertilization of the Egg results in the growing Embryo IV. Fleshy fruit forms from the rest of the Ovary and Ovule o Plant vs. Animal Plants Start with Meiosis, Animals End with Meiosis Sex Determination o Different species have different cellular sex mechanisms Homogametic – XX, same sex chromosome Heterogametic – XY, different sex chromosomes o Determination 1) Human 2) Insect Number of X Chromosomes to Autosomal Sets Ratio 1.0 = Females Ratio 0.5 = Males 3) Bird ZZ = Males ZY = Females 4) Alligators O 33 = 100% Male < 33 = 100% Female > 33 = 95% Female 5) Bonilla Worm Hits the ocean floor? Becomes Female Hits the Female? Become sperm producing machine 6) Parthenogenesis Bees, Wasps, all males are Haploid Organisms Female are all diploid o Morgan’s Work on Chromosomes Morgan wanted to experiment with Phenotypes, specifically hoping to induce some sort of mutation in his breeding flies Inducing through Radiation White eye mutation occurred and he found 44:1 ratio, with females being more red Test Cross Heterozygous Female with White Eyed normally gives 1:1, but white eyes males die before birth Chapter 10: Chromosome Organization and Molecular Structure Chromosome Overview o Functions of the Chromosome 1) Genetic Storage 2) Genetic Expression 3) rRNA Assembly 4) Compacting and Sorting (**most critical feature) o Characteristics Bacteria Circular (but Borrelia is linear…) Nucleoid Region Eukaryotes Linear Contained in Nucleus Diploid somatic cells (24 different types) Viruses o “Bad news wrapped up in a protein” o Overview Genetic material determines the shape and size of a virus Nucleic acid and proteins are present in the virus Viruses need a host cell in order to infect This infection id dependent on the Host Range Host Range is in essence the ability for the host to be infected Primary and Secondary Hosts allow for viral Emergence (passing it on)
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