FIMS Midterm Genetics 1-4.pdf

= High Genetics Session 1-4 Yield Chad Gentry (330) 413-8427 [email protected]  Basics of the Cell – Prokaryotes and Eukaryotes (Session 1- 2 Tip: Think about the difference between prokaryotes and eukaryotes a...

= High Genetics Session 1-4 Yield Chad Gentry (330) 413-8427 [email protected]  Basics of the Cell – Prokaryotes and Eukaryotes (Session 1- 2 Tip: Think about the difference between prokaryotes and eukaryotes and what that allows one to do over the other Learning Objectives 1) Define the characteristics of life and the 3 principles of cell theory. 2) Distinguish between the characteristics of prokaryotic and eukaryotic cells. 3) Recall the morphologies and arrangements of prokaryotes. 4) Define the functions of pili, capsules, biofilms, and peptidoglycan. 5) Compare and contrast the cell walls of Gram-positive and -negative bacteria. 6) Define the location and function of each of the following eukaryotic cell structures: rough endoplasmic reticulum, smooth endoplasmic reticulum, nucleus, ribosomes, nucleolus, chromatin, nuclear envelope, nuclear pore, Golgi apparatus, plasma membrane, mitochondria, lysosome, cytoskeleton (filaments and microtubules), peroxisome, centrosome, intracellular junctions, and flagella. What is life? All living things… (LO1) Principles of the cell theory (LO1) 1) Every living organism is composed of one or more cells 2) The cell is the smallest unit having the properties of life 3) Cells are produced only by the growth and division of pre-existing cells Types of Cells (LO2) Prokaryotes: No nucleus – nucleic acids are localized in an area called the nucleoid but not membrane bound Lacks membrane-bound organelles Eukaryotes: One to many membrane-bound nuclei – DNA is wound up by histone proteins Complex organelles Note: Prokaryotes became Eukaryotes  (LO3) Prokaryote Morphologies Prokaryote Arrangements Flagella (LO6): Structure used for motility (ATP required to turn the flagella) Bacteria have different organizations of flagella One flagella = monotrichous Collection of flagella in one area = lophotrichous Flagella all over the surface = peritrichous Eukaryotic flagella and cilia: (LO2) Eukaryotic flagella are used form motility Cilia can also be used for motility as well as the movement of fluids across a surface as in the mucosa of the respiratory tract Prokaryotes & Eukaryotes (LO2) Gram-positive and Gram-negative cell wall composition: (LO5) Lipoteichoic acid Teichoic acid Peptidoglycan is a thick layer exposed to Peptidoglycan is a thin layer contained in the the environment periplasm Teichoic and lipoteichoic acids present Lipopolysaccharide outer membrane (brown) Gram-Positive: Teichoic and lipoteichoic acids Functions Teichoic acids Required for the viability of Negative-charged polymers Gram-positive bacteria anchored into the peptidoglycan Provide a pathway for positively-charged Lipoteichoic acids substances through the Negatively-charged polymers complicated peptidoglycan anchored into the cell membrane network Adherence of bacteria to surfaces Gram-negative outer membrane: (LO5) Lipopolysaccharides (LPS) bind to host receptors and trigger an immune response LPS is released upon death of the bacteria, when in excess, leading to a condition called septic shock: Capillaries leak (rash), Blood vessels dilate (low blood pressure), Fever develops, Clots form at the same time platelets are depleted. This can be life threatening! Gram-negative porins: (LO5) Porins are channels found in the outer membranes of Gram- negative bacteria These structures regulate what substances can get into and out of the bacteria including antibiotics (esp those that interrupt peptidoglycan synthesis) Porin-mediated antibiotic resistance – Gram-Negative (LO5) Slight alterations of structure through mutation can limit antibiotic uptake If the structure of Gram- negative bacteria mutates, these bacteria can develop immunities to antibiotics. Functions of the Cell Organelles Tip: Think about what happens when you manipulate the function of an organelle and how their function connect to other organelles. Functions of Pili – Attachment & Communication: (LO4) Pilli Function in attachment to tissue cells or surfaces (P-pili or other designations) F-pili (or sex pili) are specialized structures that allow gene transfer among bacteria through a process called COJUNCTION! Intercellular Junction – Attachment and Communication: (LO6) Tight junctions Also known as zonula occludens Prevent loss of fluids from tissues or blood into tissues and maintains polarity of apical (luminal side) and basolateral surfaces (tissue side) Desmosomes Intermediate filaments bind to plaques anchoring cadherin proteins that interact to form attachment of two adjacent cells Gap junctions Proteins that form channels between two adjacent cells to allow molecules, ions, and electrical signals to pass between them Functions of Biofilms & Capsules – Resisting Immunity: (LO4) Biofilms Capsules Biofilms formed by aggregation Polysaccharide or protein that is secreted to cover the of bacteria secreting protein or surface of bacteria or other carbohydrate coats (capsules) organisms (some fungi) which combine to surround a Functions to prevent host colony of bacteria cells from engulfing the microorganism and These biofilms allow bacteria destroying it (ie to persist in environments phagocytosis despite body responses such Microbes expressing as immunity and chemicals capsules demonstrate a that would remove them “halo” upon staining and colonies grown on agar medias appear smooth Location & Function of Ribosomes: (LO6) Function to produce proteins in a process called translation Prokaryote ribosomes are 70S composed of a small subunit (30S) and large subunit (50S) Eukaryote ribosomes are 80S composed of a small subunit (40S) and large subunit (60S) Antibiotics can target prokaryote ribosomes somewhat specifically and inhibit protein synthesis. Deoxyribonucleic acid (DNA) DNA contains the information to produce all of the structures and enzymes of the cell Transcription produces a nucleic acid copy (mRNA, messenger RNA) of a particular cellular component Translation of the mRNA by ribosomes produces protein (structure or enzyme) Structures and organelles of Eukaryotic Cells – Plasma Membrane (Cell Membrane) (LO6) Material continually moves from outside the cell to inside and vise versa Import (endocytosis) – capture materials outside of the cell and upon entry usually fuse with lysosomes (digestion) Export (exocytosis) – moves materials to the outside of the cell Structures and organelles of Eukaryotic Cells – Outside the nucleus (LO6) Chromatin in the nucleus Chromatin = complex of DNA and proteins. It packages long DNA molecules into compact structures.(mitosis) Histones wrap the DNA to compact it to fit into the nucleus; also regulate production of cell components from DNA Nuclear Membrane (nuclear envelope) DNA is contained within the nucleus which consists of a double membrane known as the nuclear membrane The only way into and out of the nucleus is through nuclear pore complexes (NPC)  Structures in the nucleus (LO6) Nuclear pore complex (NPC) regulate the transport of proteins and DNA/RNA across the nuclear membrane Nucleolus synthesizes ribosomal RNA and forms ribosomes Centrosomes (with centrioles) (LO6) Centrosomes Microtubule organizing centers for animal cells functioning in cell division The barrel-shaped centrosomes are made up of centrioles (clusters of microtubules) Direct movement of chromosomes during cell division; centrioles create spindles that help separate the duplicated chromosomes during mitosis  Endoplasmic Reticulum (ER) (LO6) Rough ER Ribosomes with a “rough” appearance Site of protein production from mRNA (translation) Smooth ER Involved in the synthesis of fatty acids, phospholipids, and steroids Glycogen breakdown and release of glucose Detoxification involves adding –OH groups to drugs increasing solubility to be flushed from the body Golgi Apparatus (Golgi, Golgi body, Golgi complex): (LO6) Modification and synthesis of the carbohydrate portions of glycoproteins All proteins, lipids, and polysaccharides that move to the trans Golgi (distribution center) are tagged with carbohydrate or phosphate bind to the appropriate transport vesicle traffic to the lysosomes, plasma membrane, or cell exterior. Cytoskeleton (LO6) Lysosomes and Peroxisomes - Phagocytosis (LO6) Lysosomes Contains acid hydralases able to breakdown proteins, nucleic acids, carbohydrates, and lipids. Used to degrade engulfed materials as well as components of the cell itself Used by phagocytes in the digestion of invading microbes. Peroxisomes Site of oxidation reactions which can produce hydrogen peroxide. Oxidation reactions break down substances such as fatty acids providing a source of metabolic energy or ATP (adenosine triphosphate).  Mitochondria & its source of energy (ATP) (LO6) Organelles that produce ATP providing energy for cellular processes to occur Cells requiring large amounts of energy have numerous mitochondria The conversion of ATP to ADP produces energy! Nucleic Acid Structure (Session 3) Tip: Know the significance of each experiment Learning Objectives 1)Recall the setup, results, conclusions, and relevance of the experimentation carried out by: Griffith, Avery, MacLeod, McCarty, Hershey, Chase, Franklin, Wilkins, Chargaff, Watson, and Crick. 2)Identify the components of a nucleoside and nucleotide. 3)Differentiate between the structures of DNA and RNA. 4)Identify the biological significance of major and minor grooves Griffith’s Experiment (1920) – Identifying the Genetic Material (LO1) Griffith injected live or heat-killed bacteria into mice and observed for disease development and death. He was using two strains of pneumococcus: SMOOTH (type IIIS) bacteria Express a capsule Produce smooth colonies when grown on agar media ROUGH (type IIR) bacteria No capsule Produce rough colonies when grown on agar media Griffith’s Experiment (1920) – The Transformation Principle (LO1) VS VS Some “factor” from the dead (heat-killed) type IIIS bacteria transformed the type IIR bacteria into type IIIS bacteria  Avery-MacLeod-McCarty Experiment – Searching for the Transformation Principle (LO1) What substance is getting transformed from dead IIIS to live type IIIS bacteria? DNase = destroys DNA RNase = destroys RNA Protease = breaks down proteins What they found: DNA is the transformation principle  The Others Trying to Identify Genetic Material: Hershey & Chase (1952) (LO1) Studied viruses that infect bacteria (bacteriophage T2) Hypothesis = DNA is the genetic material of the T2 bacteriophage Hershey & Chase Experiment (LO1) Hershey & Chase Experiment (The Findings) (LO1) DNA component of the bacteriophages is injected into the bacterial cell while the protein component remains outside It is the injected component — DNA — that can direct the formation of new virus particles complete with protein coats DNA is the heredity factor  Determining the Structure of DNA – Franklin & Wilkins (LO1, LO3) Methods for determining the structure of DNA: 1. Ball-and-stick models First used by the chemist Linus Pauling Pauling put forward the first structure of DNA with 3 strands (1953) These models were used by Watson and Crick to discover the actual structure of DNA with 2 strands (using physics) 2. X-ray diffraction Used by Maurice Wilkins and Rosalind Franklin Exposed DNA to X-rays (1953) Exposure produces a clear diffraction pattern (can be interpreted by mathematical operation to provide information about the structure of DNA) Results Helical Contains more than 1 strand 10 base pairs per turn of the helix  Determining the Structure of DNA – Chargaff (1950) (LO1, LO3) Methods for determining the structure of DNA: DNA biochemical composition Erwin Chargaff analyzed the composition of DNA isolated from numerous species (1950) Chargaff’s findings regarding DNA: Amount of Adenine (A) = Thymine (T) Amount of Guanine (G) = Cytosine (C) Race for DNA comes to a close – Watson & Crick (1953) (LO1, LO3)  Structure of Nucleic Acids (LO2) Nucleotides = Repeating structural unit of nucleic acids Structure found in DNA and RNA Components: 5-carbon sugar Nitrogen-containing base Phosphate Two atoms (OH) shown to the right are found in single nucleotides but not when the nucleotides are incorporated into DNA or RNA Also notice that deoxyribose is missing an O at the 2’ carbon in DNA as compared to the ribose of RNA (see arrows) Structure of Nucleic Acids (LO2) Nucleoside terminology Sugar + Base o Named according to the added base and sugar ▪ Using adenine as the base below… ▪ If adenine is attached to Ribose = Adenosine ▪ If adenine is attached to Deoxyribose = Deoxyadenosine What happens if you start adding PHOSPHATE to adenosine? AMP -> ADP -> ATP (LO2, LO3) One phosphate added → adenosine monophosphate (AMP) Two phosphates added → adenosine diphosphate (ADP) Three phosphates added → adenosine triphosphate (ATP) BASE Nucleoside Adenine Adenosine Guanine Guanosine Thymine Thymidine Cytosine Cytidine Uracil Uridine Forms of DNA (LO3) 1. B-DNA (Most Common) DNA is negatively charged (phosphates) and the double helix with a right-hand twist Base pairs are perpendicular to the longitudinal axis of the DNA Most free-floating DNA in the cell and DNA in any aqueous solution or near physiological osmolarity/pH 2. A-DNA More compressed conformation of DNA First observed during in vitro crystallization of DNA May occur in RNA-DNA hybrid double helices or when the DNA is complexed to enzymes 3. Z-DNA Transient form found in GC-rich areas of DNA Twists occur in the opposite direction (left-hand twist) May occur in response to methylation of DNA (epigenetic regulation) Three Types of RNA (LO3) 1. Ribosomal RNA Part of the ribosomes to permit interaction with mRNA during translation 2. Messenger RNA (mRNA) RNA copy of a portion of DNA (gene) made during transcription mRNA is made in the nucleus and then transported to the cytoplasm for translation 3. Transfer RNA (tRNA) RNA conjugated with a specific amino acid Participates in translation by bringing the specific amino acid to a growing polypeptide chain Major & Minor Grooves of DNA (LO4) Glycosidic bonds holding each base pair are NOT directly across the helix from one another = intertwined chains then create major and minor grooves These grooves are binding sites for proteins involved in DNA replication, transcription, repair, and regulation Transcription factors and enzymes usually bind in these sites  Chromatin and Chromosomes (Sessions 4) Tip: Understand the DNA packaging (Know it cold) Learning Objectives 1) Define the following: nucleoid, chromosome, sister chromatids, kinetochore, and telomere. 2) Differentiate between euchromatin and heterochromatin. 3) Distinguish between G bands and C bands. 4) Recall the different levels of organization of DNA into chromosomes and the accompanying mechanisms of compaction into the nucleus. 5) Identify the structures making up a nucleosome and the function of nucleosomes. 6) Define the role of changes in chromatin structure. 7) Recall the mechanism of X-inactivation and its linkage to disease. 8) Define condensins and cohesins and their influence upon the structure of chromosomes. Chromosome karyotyping Chromosomal characteristics are shown as metaphase chromosomes Note: Humans have two sets of 23 Chromosomes are arranged in pairs in descending order of size and according to the chromosomes (46 total) position of the centromere (one maternal and one paternal Autosomes = all chromosomes in the genome except for the sex determining chromosomes chromosome x 23 pairs = 46) Sex chromosomes = chromosomes containing genes for sex determination For each chromosome: One maternal copy and one paternal copy Female karyotype = 46XX Male karyotype = 46XY Chromosome Structure (LO1) Sister Chromatids = Each chromosome and its duplicated version. Non-Sister Chromatids = Chromatids of homologous chromosomes. Chromatids of a chromosome are joined together at the Centromere Note: Chromatin = DNA + Protein  Brief Review of the Cell Cycle Chromosomes are duplicated during the Synthesis (S) phase of the cell cycle prior to mitosis Sister chromatids are separated during anaphase of mitosis to create two new daughter cells with the same number of chromosomes as the parent Chromosome Structure (LO1) Kinetochore Disc-shaped protein, holds the sister chromatids together Site of attachment of spindle fibers during cell division to pull the sister chromatids apart G-banding vs C-banding (LO3) Chromosome bands Q-banding (Q = quinacrine) Quinacrine fluorochrome (fluorescent microscopy) produces intense fluorescence in DNA regions rich in A-T; G-C rich areas are weakly fluorescent G-banding (G = Giemsa) Giemsa stain (light microscopy) similarly are more intense in A-T rich regions – used for chromosome abnormalities. R-banding (R = reverse) Fluorochromes that bind to GC-rich regions of DNA Can counterstain to increase intensity C-banding (C = constitutive) Identifies heterochromatin such as repetitive DNA surrounding centromeres or areas of gene silencing – only stains centromere.  Chromosome Type Based on Centromere/Sequences (LO4) Eukaryotic chromosomes 1. Unique or nonrepetitive DNA sequences o Sequences that appear once or a few times within the genome o Example = majority of protein genes o ~40% of genome 2. Moderately repetitive DNA sequences o Sequences found a few hundred to several thousand times in the genome o Example = ribosomal RNA (rRNA) o Usually associated with materials required in high amounts 3. Highly repetitive DNA sequences o Sequences found tens of thousands to millions of times in the genome o Usually short in length o Example – Alu sequences (10% of genome) are sequences that can be copied and inserted into any region of the genome; thought to drive evolutionary changes in proteins  Ends of Eukaryotic Chromosomes (LO1) Telomeres Two regions of noncoding DNA are the centromere and telomere Telomeres are the ends of chromosomes. They are long! Up to 2,000 repeats of the sequence 5’-TTAGGG- 3’ Prevent ends of chromosomes from accidentally becoming attached to each other With each replication of the cell the telomere is shortened imposing a finite life span on cells.  Packaging of DNA into the Nucleus – Making the Nucleosome (LO4, LO5) Step 1: Histone Segments of linear DNA (~146 bp) wrap around an octamer of histone proteins (core histones) to form nucleosomes Histone Octamer (2 of each): H2A, H2B, H3, H4 It’s the Arginine's of the histone proteins that interact with DNA phosphate. What we end up getting = Nucleosome!!! “beads on a string”  Packaging even Tighter – H1 Histones (LO4) Step 2: H1 histones help further compact adjacent nucleosomes. Nonhistone proteins bind in the linker DNA region of chromatin and aid. Once Nucleosomes associate, they form more compact structure (shortens 7-fold: 30 nm diameter) Our Two 30nm DNA Models 30-nm fibers interact with the nuclear matrix to form radial loop domains  Radial Loop Domains (LO4) Step 3: DNA-bound proteins bind to sequences in the DNA found at regular intervals throughout the genome Radial Loop Domains (below) are formed by binding DNA with nuclear matrix Not only does the formation of radial loops aid in compacting the DNA it also organizes the chromosomes into discrete locations within the nucleus This permits efficient gene expression and regulation, chromosome separation during mitosis, and DNA replication Why Radial Loops? What about Mitosis? (LO2, LO4, LO6) Radial loops are further compacted into heterochromatin or euchromatin Heterochromatin Tightly compacted regions of chromosomes (cannot observe radial loops) Transcriptionally inactive By the end of prophase and entry into metaphase, the replicated chromosomes (sister chromatids) are entirely heterochromatic (we want our chromosomes sorted) Euchromatin Less condensed regions of chromosomes (radial loops evident) Capable of transcription Most chromosomal DNA is found in this state during interphase  Condensin and Cohesin (LO6, LO8): Structural Maintenance of Chromosome (SMC) proteins use ATP to alter chromosome structure. Two Types: 1. Condensin (SMC2 + SMC4 + CAP-H + CAP-D + CAP-G) = coats chromatids leading to conversion of euchromatin -> heterochromatin (just before mitosis) 2. Cohesin (SMC1 + SMC3 + Scc1 + Scc3) = promotes binding of sister chromatids after S phase until late prophase. Removed by anaphase promoting complex during anaphase (when chromatids are separated). Summary of DNA Packing: X-Inactivation (LO7): Normal protein production (gene activity) by just one of the X chromosomes is normal for humans. Women have 2 X chromosomes but one is silenced through a process termed X-inactivation The inactive X chromosome is compacted tightly into a structure known as a Barr body That Barr body will always be the same X chromosome (does not change through life).  X-inactivation & Diseases (LO7): Aneuploidy = extra or missing chromosome Ex: X-inactivation silencing extra X chromosome to some degree. Triple X syndrome Women with XXX genotype (1 in 1,000 female newborns) Normal birth, female sex characteristics, and able to have children Learning difficulties, late development of motor skills in infancy, and problems with muscle tone Klinefelter syndrome Male with XXY genotype (1 in 500-1,000 male newborns); possible more than 2 X chromosomes Infertile men, reduce muscle strength, enlarged breast, tall X-inactivation & Diseases – What the Calico (LO7): If a female cat is heterozygous for black and tan coat color gene found on chromosome X – she will inactivate both X at random in different cells during development. Result is an alternating patch of black and tan fur due to parts of black X chromosome and parts of tan X chromosome inactive (Not all of either): Human females can experience similar activity (not as obvious as in Calico Cats) THANK YOU SO MUCH!!!! Chad Gentry (330) 413-8427 [email protected]