Cell cycle_Cloning_F23.pdf

Cell Cycle and Growth Control – Ch 30 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 1 Learning Objectives By the end of this lecture, students will be able to: • Characterize and identify phases of the cell division cycle (cell cycle) • Characterize and id...

Cell Cycle and Growth Control – Ch 30 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 1 Learning Objectives By the end of this lecture, students will be able to: • Characterize and identify phases of the cell division cycle (cell cycle) • Characterize and identify the stages of mitosis • Define and identify the roles of the different cyclins, cdk’s, and cki’s • Compare and contrast the characteristics of apoptosis and necrosis 2 Cell Cycle • If an organism requires additional cells for growth or to replace lost cells, new cells must be produced by cell division (proliferation) • Somatic cells generate by division of existing cells • Contents are duplicated and then divided to produce two identical daughter cells 3 Cell Cycle Life cycle of the cell • Interphase: an extended period between cell divisions, DNA synthesis, and chromosome replication phase • • M phase (mitotic phase): division of genetic information • • Subdivided into three phases: G1, S phase, and G2 Divided into five phases: prophase, prometaphase, metaphase, anaphase, and telophase Cytokinesis: separation into two distinct daughter cells that enter interphase 4 Interphase - Overview G1, S, G2 • G1: growth; proteins necessary for cell division synthesized • G1/S checkpoint: regulated decision point • S: DNA synthesis • G2: preparation for cell division • G2/M checkpoint: only passed if DNA is completely replicated and undamaged 5 G1 and G0 phases G1 phase: Growth phase and preparation time for DNA synthesis in S phase. • RNA and protein synthesis • Organelles and intracellular structures duplicated and cell grows • Length varies among cell types • Mature cells permanently in G1 G0: Cells in G1 that are NOT committed to DNA synthesis  resting state • Inactive or quiescent cells may reenter active cell cycle with proper stimulation Restriction point: cells passing this point are committed to DNA synthesis in S 6 S phase • Synthesis of nuclear DNA (DNA replication) • 46 chromosomes are copied to form sister chromatid • ATP-dependent unwinding of DNA by helicase exposes binding site for DNA polymerase to synthesize new DNA • Multiple replication forks are active to ensure entire genome is duplicated within time span of S • At the end of DNA synthesis, chromosome strands are condensed into heterochromatin (tightly coiled/condensed) • Time for completion constant across cell types: ~ 6 hours 7 G2 phase • Gap between completion of S and start of mitosis • Preparation time for nuclear division • Safety gap, ensures DNA synthesis is complete • Checkpoint: • • Intracellular regulatory molecules asses nuclear integrity Phase lasts ~ 4 hours 8 M Phase - Overview • Mitosis (nuclear division): • • • • Continuous process divided into 5 phases Separation of sister chromatids ~1 hour Cytokinesis: • • Separation of cytoplasm Formation of two separate daughter cells from one parent cell 9 Mitosis Prophase • Nuclear envelope is intact while duplicated chromatin from S phase condenses into defined chromosomal structures called chromatids • Two chromatids are connected at a centromere • Kinetochores form and associate with each chromatid • Nucleolus (organelle within nucleus) disassembles 10 Mitosis Prometaphase • Disassembly of nuclear envelope • Spindle microtubules bind to kinetochores • Chromosomes pulled by microtubules of the spindle 11 Mitosis Metaphase • Chromatids align at the equator of the spindle, halfway between two poles • Forms the metaphase plate 12 Mitosis Anaphase • Mitotic poles are pushed apart as polar microtubules elongate • Centromere splits into two • Paired kinetochores separate • Sister chromatids migrate to opposite poles of spindle 13 Mitosis Telophase • Kinetochore microtubule disassembly • Mitotic spindle disassociation • Nuclear envelope forms around the two nuclei containing chromatids • Chromatids decondense • Nucleoli reform 14 Cytokinesis • Completion of cell cycle • Cytoplasmic division creates separate daughter cells • Actin microfilament ring forms machinery needed • Contraction of actin-based structure forms cleavage furrow • Furrow deepens until opposing edges meet • Plasma membranes fuse 15 Genetic Consequences of the Cell Cycle • Cycle produces two cells that are genetically identical to each other. • Newly formed cells contain a full complement of chromosomes. • Each newly formed cell contains approximately half the cytoplasm and organelle content of the original parental cell. 16 Regulation of the Cell Cycle 17 Cell cycle regulators • Control cell cycle progression • Expression of proteins and enzymes depends on cell cycle phase • Cell cycle mediators classified as: • • Cyclins Cyclin-dependent kinases (CDKs) • Complexes of cyclins and CDKs possess enzymatic activity • Cyclin-dependent kinase inhibitors (CKI) can be recruited to inhibit cyclin-CDK complexes 18 Cyclins • Categorized as cyclin D, E, A, B • Different cyclins expressed to regulate specific phases • Concentrations rise and fall through cell cycle due to synthesis and degradation • D type: G1 phase regulators (progression through restriction point) • E and A type: S phase regulators • B and A type: mitotic phase regulators 19 Cyclin-dependent kinases • CDK levels stay constant • Enzyme activity changes depending on concentration of cyclins (required for activation) • Cyclin-CDK complex is phosphorylated • Complex phosphorylates substrate proteins on serine and threonine • Changes activation status of substrate proteins • Active CDK2 activates target proteins in S phase transition and DNA synthesis • Active CDK1 targets activated proteins to initiate mitosis 20 Checkpoint Regulation • Checkpoints monitor the completion of critical events • Delay progression to next phase/step if necessary • Cell depends on stimuli from growth factors to progress through cell cycle prior to restriction point • After restriction point, cell does not require further stimuli 21 Tumor Suppressors and checkpoints • Function to halt cell cycle progression within G1 • Can’t go past restriction point • Occurs if cell growth is not needed OR undesirable OR DNA is damaged • G1 checkpoint: ensure G1 completed before starting S phase 22 Tumor Suppressors and checkpoints Retinoblastoma (RB) protein: halts cell in G1. • Resting cells: RB has few phosphorylated aa residues • • Prevents entry into S by binding to transcription factor E2F and binding partner DP1/2 (critical for G1/S transition) Cycling cells: RB is hyperphosphorylated due to growth factor stimulation and signaling via MAP kinase cascade • • • • Cyclin D-CDK4/6 is activated and phosphorylates RB Cycle E-CDK2 phosphorylates RB, allowing cell to move out of G1 Hyperphosphorylated RB can’t inhibit E2F binding to DNA E2F binds DNA and activates genes for S phase 23 24 Tumor Suppressors and checkpoints p53 • Major regulatory role in G1 • Nuclear DNA damage  phosphorylation, stabilization and activation of p53 • Activated p53 stimulates CKI transcription to produce protein p21 • p21  Halts cell cycle progression and allow DNA repair • If damage is irreparable, p53 triggers apoptosis 25 Tumor Suppressors and checkpoints Cyclin-dependent kinase inhibitors (CKI) • Two classes exist: 1. INK4A: inhibit D type cyclins from associating with and activating CDK 4 and 6 2. CIP/KIP: potent inhibitors of CDK2 kinases • Includes p21 26 Tumor Suppressors and checkpoints • G2 checkpoint: occurs after S; important for mitosis to not begin before DNA is duplicated during S • CDK1 activity: controls entry into mitosis. • • • During G1, S, and into G2, CDK1 is phosphorylated on tyrosine residues = inactive Phosphorylations must be removed for CDK1 to bind cyclin B to stimulate progression through cell cycle Cdc25c phosphatase: removes inhibitory phosphorylations from CDK1 • Allows active CDK1-cyclin B complex to move to nucleus and activate key components of mitosis (microtubules) 27 Cell Death Necrosis and Apoptosis 28 Necrosis • Passive, pathological process induced by acute injury or disease • Group of cells in the same region of a tissue undergo necrosis at the same time after an insult • Cells increase in volume and lyse (burst) • Release intracellular contents • Induces a potentially damaging inflammatory response • Process completed within several days 29 Apoptosis • Process of programmed cell death • Cells deprived of survival factors activate intracellular suicide program and die • Apoptotic cells shrink in size and do not lyse • Plasma membrane remains intact but portions bleb, or bud off • Lose their asymmetry and ability to attach to neighboring cells • Membrane phospholipid, phosphatidylserine (normally present on inner membrane toward cytosol) becomes exposed on cell’s surface • Chromatin of apoptotic cells segment and condense • Mitochondria release cytochrome c (active, ATP requiring process) 30 Apoptosis • Apoptotic cells are engulfed by phagocytic cells, macrophages, and dendritic cells by binding to exposed phosphatidylserine • Macrophage internalizes and degrades cell, reduces risk of inflammation from cell death • Phagocytic cells release cytokines (IL-10 and TGFbeta) to inhibit inflammation • No extensive damage done to neighboring cells • Process completed within a few hours 31 Cloning and Biotechnology – Ch 34 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 32 Learning Objectives By the end of this lecture, students will be able to: • Describe the importance and function of restriction enzymes • Explain how recombinant DNA is made and the basics of cloning • Identify the features of cloning vectors • Characterize the thermocycling steps of PCR • Identify and describe the techniques used to analyze DNA, RNA, proteins 33 Recombinant DNA Technology 34 Recombinant DNA technology (Genetic Engineering) • Techniques to accurately and efficiently cleave DNA helped develop this technology • Set of techniques for locating, isolating, altering, and studying DNA segments • Goal = combine DNA from two distinct sources • Recombine any DNA sequence from any source 35 Restriction Enzymes • Discovered in late 1960’s • Restriction endonucleases • Recognize specific nucleotide sequences in DNA to make double stranded cuts • Restriction sites: specific site on DNA where cuts are made 36 Restriction Enzymes • Produced naturally by bacteria as defense against viruses 37 Restriction Enzymes • Several types have been isolated (~800) • Vary in nucleotide sequence and length of recognition site • Type II restriction enzymes are most commonly used in molecular genetics • Commercially available • Name signifies bacterial origin 38 Restriction Sites • Recognition Sequence • 4 to 8 bp long • Palindromic – read the same in 5’ to 3’ direction on the two complementary DNA strands 39 Type of Fragment End Produced Cohesive ends: 1. Staggered cut in the DNA • fragments with short, single-stranded overhanging ends • Can spontaneously base pair to connect fragment • “Sticky ends” • Blunt ends: 2. even-length ends from both single strands • Cuts in the middle of recognition site • 40 Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends. Note: Blunt end fragments are joined by a different mechanism (bacteriophage T4 ligase) 41 DNA Cloning 42 DNA Cloning • Gene cloning: amplifying a specific piece of DNA via a bacteria cell • Introduce foreign DNA molecule into replicating cells produces many identical copies DNA is first cleaved by restriction enzymes 1. 1. 2. Creates many fragments (100-1,000) DNA fragments are joined to DNA vector molecule – cloning vector 43 DNA Cloning • Cloning vector: a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell • Cloning vector with inserted DNA fragment is added to a single host cell (bacteria) to be replicated • As host cell multiplies, forms a clone where every bacterium carries copies of the same inserted DNA fragment • Amplified cloned DNA fragment released from vector using restriction enzymes and isolated 44 Cloning Vector Properties 45 Bacterial Plasmids Prokaryotes have single, large, circular chromosomes AND some have plasmids • Plasmids: small, circular, extrachromosomal DNA molecules from bacteria • • Contains “special” genes that are not related to basic life functions • • i.e. antibiotic resistance Plasmid replication may or may not occur at the same time as chromosomal division Can be transferred from one bacterial cell to another • Plasmids can be easily isolated from bacterial cells • 46 A foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes. Transformation: process of introducing foreign DNA into bacteria or yeast cell. 47 48 Gene Libraries • DNA library: a collection of clones containing all the DNA fragments from one source 1. Genomic DNA library: digestion of the total DNA with a restriction enzyme and subsequent ligation to a vector 2. cDNA libraries: consisting only of those DNA sequences that are transcribed into mRNA 49 Genomic DNA library: digestion of the total DNA with a restriction enzyme and subsequent ligation to a vector 50 cDNA libraries: consisting only of those DNA sequences that are transcribed into mRNA 51 Sequencing of Cloned Fragments • Base sequence of cloned DNA fragments can be determined • Originally done by using Sanger dideoxy method 1. ssDNA used as a template for DNA synthesis by DNA polymerase 2. Radiolabeled primer complimentary to 3’end of target DNA is added 3. 4 dNTPs are added (dATP, dTTP, dCTP, dGTP) 4. Divided into 4 reaction tubes with small amounts of only one type of ddNTP (no 3’ hydroxyl group, terminates elongation) 5. Results in DNA strands of different lengths, each terminating at a specific base 52 53 SOUTHERN BLOTTING 54 Southern Blotting • Technique used to detect mutations in DNA • Combines use of restriction enzymes, electrophoresis, and DNA probes 1. Extract DNA 2. Cut DNA (restriction enzymes) 3. Separate DNA (electrophoresis) 4. DNA excised from gel and denatured (double strands are separated) 5. Blotted on membrane with a probe (complimentary DNA) to detect DNA of interest 55 56 Polymerase Chain Reaction 57 Polymerase Chain Reaction (PCR) • Test tube method of amplifying selected DNA sequences • Amplifies DNA sequence of interest exponentially (millions of copies) in a few hours by using specific primers • Uses thermostable DNA polymerase to amplify targeted portions of DNA • Each cycle doubles amount of DNA – exponential increase • Amplified sequence is analyzed by gel electrophoresis, southern blotting, or direct sequencing 58 Steps in one cycle of PCR 59 60 Analysis of Gene Expression 61 Determination of mRNA levels • Hybridization of labeled probes to mRNA or cDNA produced from mRNA 1. Northern Blots 2. Microarrays 62 Northern Blots • Method is similar to southern blot • Sample contains mRNA • Separated by electrophoresis • Transferred to membrane • Hybridized to radiolabeled probe specific for target mRNA • Bands analyzed by autoradiography • Indicates amount (strength of band) and size (position on membrane) of particular mRNA in sample 63 Microarray • Contain thousand of immobilized DNA sequences organized in a very small area Gene Chip – small glass slide or membrane containing thousands of tiny spots of DNA • Each spot corresponds to a different gene • • Used to detect gene variations or mutations in DNA • Used to determine patterns of mRNA production • • Gene expression analysis Can analyze thousands of genes at the same time 64 Microarray • mRNA is converted to cDNA and labeled with a fluorescent tag • Amount of fluorescence at each spot = amount of mRNA in the sample • Determine differing patterns of gene expression in different types of cells 65 Analysis of Proteins 66 Enzyme-linked immunosorbent assays (ELISA) • Performed in wells of a plastic microtiter plate • Antigen is bound to plastic of dish • Probe has antibody specific for protein being measured • Antibody is bound to an enzyme to allow for detection • • Produce color product when enzyme is exposed to substrate Amount of color = amount of protein in the sample 67 Western Blots • Immunoblots • Method is similar to southern blot • Sample contains protein • Separated by electrophoresis • Transferred to membrane • Probe is labeled antibody 68 Summary of techniques used to analyze DNA, RNA, and proteins 69