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40 Cards in this Set
- Front
- Back
What is the Cell cycle?
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Interphase + Mitosis + Cytokinesis
Interphase: G1: -transition from M to S phase -synthesis of proteins that be required during S phase -G0 = cells that become arrested in G1 (quiescence) -check for cell size, nutrients, growth factors (D-type cyclins), and DNA damage S: -synthesis phase -DNA synthesis: double amount of DNA G2: -transition from S to M -synthesis of proteins need in M -check for cell size and DNA damage Mitosis: -Prophase -Metaphase -Anaphase -Telophase -chromosome segregation, cytoplasmic division -check for chromosome attachment to spindle Cytokinesis: -cytoplasmic divisions after mitosis -SEPARATE than mitosis + interphase CDKs: -cyclin dependant kinases -specific enzymes present during G1, S, G2, M -activated kinase adds phosphate to proteins CKI: -cyclin dependant kinase inhibitors -blocks action of kinases |
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What are the association of the different CDKs?
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G1:
-Cyclin D with CDk4 -Cyclin D with Cdk6 S: -Cyclin E with CDk2 G2: -Cyclin A with CDk2 M: -Cyclin B with Cde2 |
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What happens in the G1 phase?
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1. growth phase and prep time for DNA synthesis of S phase
2. RNA and protein synthesis 3. organelles and intracellular structures are duplicated and cell grows during this phase 4. LENGTH OF G1 MOST VARIABLE 5. rapidly dividing cells such as embryonic cells spend little time in G1 6. Mature cells remain permanently in G1 7. cells not committed to DNA synthesis = G0 phase 8. Restriction point in G 9. If passed through restriction can go into S phase |
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What happens in the S Phase?
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1. DNA replication occurs
2. 46 chromosomes copied forming sister chromatids 3. DNA helicase (ATP dependant) unwinds DNA 4. Mulitiple replication forks activated on each chromosome 5. entire genome duplicated during time span of S phase 6. Chromosome strands go into tightly coiled heterochromatin when DNA synthesis complete 7. Constant completion time among cell types 8. Actively dividng spend 6 hrs in S phase |
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What happens in the G2 Phase?
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1. S to M
2. Prep time for nuclear division of mitosis 3. safety gap makes sure DNA synthesis complete before M 4. checkpoint for nuclear integrity 5. lasts 4 hrs |
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What are Aurora Kinases?
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-discovered in eggs of toad
1. family of serine/threonine kinases 2. play important functional role during mitosis 3. control chromatid segregation Aurora A: -functions in prophase -critical for proper formation of mitotic spindle -recruitment of proteins to stabilize centrosomal microtubules Aurora B: -attaches mitotic spindle to centromere -cleavage furrow formation during cytokinesis Aurora C: -expressed in germ line cells, unclear function Elevated A B C = human tumors Inihibition of aurora kinases being investigated as anticancer therapies |
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What happens in the different phases of Mitosis?
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Prophase:
-condensed chromosomes start to become visible -centrosomes move apart and nucleolus starts disappearing Prometaphase: -nuclear membrane breaks down -kinetochores accessible to microtubules Metaphase: -chromosomes maximally condensed and align at metaphase plate Anaphase: -2 sister chromatids separate and move to opposite poles Telophase: -nucleus reassembly occurs, cytokinesis occurs |
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What is the Karotype test?
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test to identify and evaluate size, shape, and number of chromosomes in a sample
-extra missing or abnormal positions can be detected -samples arranged in prometaphase stage |
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What is responsible for chromosome movement during mitosis and how?
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Mitotic spindle is responsible
spindle assembly and chromosome attachment: -kinetochores - located on opposite sides of a chromosome and anchor kinetochore microtubules -centromere - ensures delivery of one copy to each daughter -unattached kinetochores - signal for mitotic checkpoint, arrest mitosis until all kinetochores correctly attached to spindle microtubules; preventing loss of a chromosome (aneuploidy) |
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What does Cytokinesis do?
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divides the Cytoplasm
-driven by actin and myosin -large organelles (ER, golgi) fragment into smaller vesicles in mitosis and reassemble in daughter cells |
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What provides evidence for control molecules in the cell cycle? What are these control molecules and how were they identified?
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Cell fusion experiments provide evidence
Mitosis-promoting factor (MPF): -moves cells through G2 checkpoint -phosphorylates lamin to break up nuclear membrane -phosphorylates condensin which triggers chromosome condensation -mitotic spindle formation -targeted protein degradation -during development of a frog oocyte, cell cycle arrested in G2 until hormone stimulates meiosis Identified by: cell cycle mutants: -temperature sensitive -PEG -used to fuse cell |
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How is Mitotic Cdk-cyclin Complex activated?
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1. MPF is inactive
2. An inhibiting kinase add 2 phosphate group to the complex 3. An activating kinase phosphorylates a third site on the complex 4. A phosphatase removes the inhibiting phosphate groups, converting complex into a single phosphorylated form = active MPF 5. Active MPF --> more active MPF 6. Activation of MPF is autocatytic |
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Who won a nobel prize for discoveries of key regulators of cell cycle and who discovered that MPF is autocatalytic?
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MPF is autocatalytic = Dr. Masui
Nobel prize = Hunt, Hartwell, and Nurse |
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What happens in Dissolution of the Nuclear Lamina, Breakdown of the Nuclear Membrane, and Re-formation of the nuclear envelope?
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Dissolution of nuclear lamina:
-at mitosis Cdc2 and other protein kinases phosphorylate lamins, causing filaments to dissociate into free lamin dimers Breakdown of nuclear membrane: -nuclear membrane fragments into vesicles -B-type lamins remain bound to these vesicles, while lamins A and C released as free dimers Re-formation of nuclear envelope: -binding of membrane vesicles to chromosomes, mediated by integral membrane proteins and B-type lamins -vesicles fuse, nuclear lamina reassembles, chromosomes decondense |
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How is the G1 restriction point controlled?
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G1 Cdk-cyclin complex does this by phosphorylating Rb protein (pRb):
-unphosphorylated pRb interacts with transcription factor E2F -this prevents E2F from stimulating transcription -G1 Cdk-cyclin phosphorylates pRb causing it to be released from E2F and allowing E2F to stimulate transcription (can be altered by p53) -once cell crosses restriction point, entry into S phase is ensured pRb = retinoblastoma protein (tumor suppressor protein) E2F = family of transcription factors involved in cell cycle regulation and DNA synthesis in mammalian cells |
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How is the spindle assembly checkpoint controlled?
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Mitotic Cdk-cyclin complex (MPF) activates anaphase-promoting complex:
-anaphase-promoting complex is a ubiqutin ligase that marks proteins for destruction by proteosomes -triggers breakdown of securin which normally binds and inhibits separase -when separase is released it targets and cleaves cohesins which causes sister chromatids to separate from each other -breaks down MPF cyclin to end mitosis -APC is not active until all chromatids are linked to spindle, actively controlled by kinetochores which bind Mad and Bub proteins when kinetochores unattached -Mad and Bub inhibit APC by controlling Cdc20 preventing anaphase |
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What are Ubiquitin and Proteosomes?
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Ubiquitin:
-target proteins for degradation of proteasomes -substrate recognition protein E3 recognize N-terminal sequence of their targets but exception are degrons (allow proteins to be ubiquitin marked) -Anaphase promoting complex functions as E3 AND binds proteins containing degrons Proteosome: -predominant protease in cytosol |
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What are the 3 major regulatory pathways?
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1. Spindle Checkpoint:
-Mad and Bubs control Cdc20 and thus control APC 2. DNA replication checkpoint: -G2 to M is blocked until DNA duplicated -final dephosphorylation step activating MPF is blocked until DNA replcated 3. DNA damage checkpoint: -can halt cell cycle at G1, S, or G2 -p53 activated by ATM-PK -p21 and PUMA (p53 Upregulated Modulator of Apoptosis) up-regulated -Mdm2 (Murine Double Minute) involved in degradation of p53 -apoptosis if too much damage |
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What are the different growth factors?
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Stimulatory growth factors:
Activate Ras Pathway: -Ras mutations found in pancreatic, colon, lung, and bladder cancers (25-30 % of cancers) Activate PI3 kinase-Akt pathway: -PTEN mutation found in 50 % prostate cancer, 35 % uterine cancer Inhibitory growth factors: -act through Cdk inhibitors -TGF beta = causes increase in Cdk inhibitor p15 and p21 -p21 plays a major role in preventing damaged DNA from passing G1 checkpoint |
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What is the PI3-kinase Akt Pathway?
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1. activation of PI 3-kinase by phosphorylated tyrosines found in receptors that have been stimulated by growth factor
2. Activated PI 3-kinase adds a phosphate to PIP2 --> PIP3 3. PIP3 recruits protein kinases to inner surface of plasma membrane leading to phosphorylation and activation of protein kinase Akt 4. Akt suppresses apoptosis and inhibits cell-cycle arrest, promoting cell survival and proliferation 5. pathway is inhibited by PTEN which catalyzes PIP3 --> PIP2 |
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What is Equilibrium Density Centrifugation?
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-showed that DNA replication is semiconservative
-technique was used to distinguish between N15 DNA (Heavy) and N14 DNA (light) -the density that came up was in between the two DNA strands showing that DNA is semiconservative |
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How is DNA replication in circular DNA?
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it has bidirectional replication forks from a single origin
this theta replication occurs in bacteria, mitochondria, chloroplasts, and some viruses |
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How is DNA replication in Eukaryotes?
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involves many replicons
replicons - units of replication of linear DNA replication starts at Autonomously Replicating Sequence (ARS) which is rich in AT |
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What is the pre-replication complex?
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Origin Recognition Complex (ORC) binds to replication origin first
MCM complex, helicase, and primase complex bind next (require assistance from helicase loaders) Pre-replication complex needs polymerase to be completed Phosphorylation of MCMs initiates replication |
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What are the main type of Eukaryotic and Prokaryotic DNA Polymerases?
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Prokaryotic:
-I, II, II, IV, V -DNA polymerase II is the primary one Eukaryotic: -a. d. e -g is found ONLY in mitochondria -Polymerase a is involved in initial synthesis of DNA strands off RNA primers -Polymerase d(lagging) and e(leading) primary polymerases, one for each strand |
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What are all the DNA replication proteins in Prokaryotes?
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DNA Polymerase I:
-proofreading, removes and replaces RNA primers DNA Polymerase III: -synthesis of both DNA strands -main one DNA Gyrase: -Type II DNA topoisomerase which relaxes supercoiling |
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What are all the DNA replication proteins in Eukaryotes?
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DNA polymerase alpha:
-complexes with primase and begins DNA synthesis DNA Polymerase gamma: -mitochondrial DNA synthesis DNA Polymerase delta and epsilon: -lagging and leading strand synthesis (also DNA repair) Telomerase: -uses integral RNA molecule as template and synthesizes DNA for extension |
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What are the DNA replication proteins in both Eukaryotes and Prokaryotes?
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Primase:
-RNA synthesis DNA Helicase: -unwinds double stranded DNA -requires ATP and breaks H bonds Single-stranded DNA binding protein (SSB): -binds to single stranded DNA DNA Topoisomerase Type I or II: -Type I makes single stranded cut -Type II makes double stranded cut -relaxes DNA supercoiling DNA Ligase: -joins together adjacent DNA strands Initiator proteins: -binds to origin of replication and begins unwinding of DNA double helix |
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What does DNA synthesis require?
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1. DNA polymerase requires a RNA PRIMER for DNA synthesis
2. Primer is a small piece of RNA, synthesized by DNA primase (10 base pairs, in bacteria = primosome, in eukaryotes = DNA Polymerase alpha) 3. Once a small piece of RNA is synthesized, DNA polymerase III will begin to add deoxyribonucleotides to the end of the RNA 4. Later during replication, DNA Polymerase I will come along and remove the RNA, replacing the RNA bases with deoxyribonucleotides |
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What are Okasaki Fragments?
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For lagging strand DNA must be read in the 3' to 5' direction
Since DNA Polymerase can only synthesize DNA in the 5' to 3' direction the DNA must be synthesized in short fragments These short fragments are Okazaki fragmnents DNA ligase seals these okazaki fragments together |
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Why is Telomerase so important?
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Telomeres are at the end of LINEAR DNA and their segments are too small to work on
Telomerase is a protein and an RNA, the RNA has a complementary sequence to the telomere and the protein has polymerase activity so it fills the gap and adds bends and BPs to form a loop and protect the DNA from being damaged Telomerase also has a capping protein thus one strand is always longer than the other found in germ and tumor cells but not differentiated cells |
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What is an Oncogene and an example?
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Oncogene:
-modified gene that codes for a protein and is believed to cause cancer -genetic mutations resulting in the activation of oncogenes increase the chance that a normal cell will develop into a tumor cell proto-oncogene = normal gene that can become an oncogene due to mutations Telomerase is an example of an oncogene: -cell loses its ability to divide because telomeres are not added to the end of chromosomes |
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Who won the Nobel prize for discovery of chromosome protection by telomeres?
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Blackburn, Greider, and Szostak
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What does DNA licensing mean?
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makes sure DNA is replicated only once
Regulation: -Cdk blocks re-licensing by phosphorylating and inactivating ORC, MCM, and helicase loaders -MCM complex helps initiate DNA replication and is displaced in the process -Geminin blocks binding of MCM complex to DNA |
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What is Blood Syndrome?
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DNA Helicase is defective causing problem in DNA replication
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What are drugs that target bacterial replication?
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Quinolone:
-target bacterial enzyme gyrase (equivalent to mammalian topisomerases) which break the phosphodiester backbone to relieve torsional strain as DNA helix unwinds to allow replication -quinolone blocks this and inhibits bacterial DNA replication -does not affect eukaryotes because topoisomerases are not affected |
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What is A-amanitin?
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a toxin that is a RNA polymerase II inhibitor
blocks transcription of single-copy gene a-amanitin has no effect on telomerase or any type of DNA polymerase |
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What are the types of DNA damage and how are they repaired?
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1.
Damage = Nicks in DNA strand Repair = DNA ligase 2. Damage = Mismatched bases Repair = excision and resynthesis 3. Damage = Uracil in DNA Repair = removed by DNA uracil glycosylase 4. Damage = Apurinic or apyrimidinc site Repair = AP endonuclease 5. Damage = Pyrimidine dimers (UV) Repair = enzymatically reversed 6. Damage = region of DNA Repair = Excision repair |
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What are ways to repair abnormal nucleotides?
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Translesional synthesis:
-repair DURING REPLICATION -DNA polymerase eta can catalyze DNA synthesis across regions with thymine dimers Excision repair pathways: POST REPLICATION -Base excision = repair single damaged bases -Nucleotide Excision Repair (NER): endonuclease -pathway that corrects thymine dimers (Xeroderma pigmentosum - incapacitated NER mechanism, cannot tolerate sunlight) Mismatch Repair: -corrects mutations that involve noncomplementary base pairs -Hereditary nonpolyposis colon cancer (HNPCC) |
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What are Mutation hot spots and what is a disease associated with it?
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30 % of point mutations involve C-->T transitions
CpG islands represent a true hot spot because cytosine is frequently methylated at position 5 Rett Syndrome: -X linked dominant, only females -mutation in gene encoding methyl-CpG-binding protein 2 (MECP2) |