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119 Cards in this Set
- Front
- Back
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Define the eukaryotic cell cycle
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PhasE G1: Cell growth; S: Synthesis; G2: growth and error checking
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Mention Chargaff's rules
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Strands are antiparallel, complementary and the total % of purines = total % of pirimidines
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Mechanism of action: Daunorubicin, doxorubicin
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Intercalate between bases of DNA interfering with activity of topoisomerase II, preventing replication
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What is hybridization; Describe denaturing Vs. renaturing
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Heat, UV light and chmicals are used to denature DNA. Hybridization is when a probe DNA binds to denatured target DNA sequences of sufficient complimentarity.
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Function of topoisomerases in prokaryotes
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Introduce negative or positive supercoiling by breaking and resealing the sugar-phosphate backbone
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Define: Histones, nucleosomes, chromatin
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Histones are rich in lysine and arginine which confers them positivity; Histones form octamers: H2A, H2B, H3, H4; Nucleosome is DNA + histone octamers linked by Histone 1 (H1)
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Euchromatin Vs. Heterochromatin
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euchromatin = loose, active; heterochromatin = condensed, inactive
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What are chromatin-modifying activities
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Histone acetylation, phosphorilation lessens their positive charge and decreases affinity for DNA
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Define: polymerase; exonuclease; endonuclease
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Polymerase: form phosphodiester bonds to synthesize nucleic acids; Exonuclease: remove nucleotides from 5' or 3' ends; Endonuclease: cut within nucleic acid and release nucleosides
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What is an RNA primer?
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Sequence required for DNA synthesis. Has 5'-3' direction.
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Mention the steps of DNA replication in prokaryotes
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1. DNA A protein recognizes origin sequence; 2. Helicase unwinds by breaking H bonds; 3. SSB prevents reassociation of loose strands; 4. Primase synthesizes RNA primer; 5. DNA pol III synthesizes DNA in 5'-3' direction (Okazaki fragments); 6. DNA pol I removes primers; 7. DNA ligasse seals Okazaki fragments; 8. DNA Gyrase (topo I) inserts negative supercoiling.
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Define : Reverse transcriptase and AZT
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It's an RNA dependant DNA polymerase. AZT might be used as a substrate and gets inserted at the 3' end, terminating replication
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DNA repair: Thymine dimers
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1. UV light creates thymine dimers during G1; 2. Removed by excision endonuclease which is defficient in XP; 3. Patched b topoisomerase and ligase.
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DNA repair: Mismatch repair
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1. During G2 phase; 2. Produced by mutations of MSH2 & MLH1 genes; 3. Associated with HNPCRC
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DNA repair: p53
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Prevents cell with damaged DNA to enter S phase
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ATM Gene
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Encodes kinase essential for p53 activity. Inactivated in Ataxia-Telangiectasia
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What are the 6 types of RNA
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1. rRNA: structural component of ribosome; 2. tRNA: carries amino acids to ribosome; 3. mRNA: contain info for AA sequence; 4. hnRNA precursor of mRNA; 5. snRNA: splices mRNA; 6. Ribosymes: RNA molecules w/enzymatic activity
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Define: promoter region
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The binding site for RNA polymerase
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Define: antitemplate (coding) strand Vs. Template strand
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The antitemplate is not used during transcription but its identical to RNA molecule, except that it has U instead of T. RNA polymerase uses template strand 3'-5' to create antiparallel and complimentary 5'-3' strand
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What is the direction of translation of the ribosome?
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reads mRNA 5'-3'; Amino to Carboxyl group.
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What is the direction of transcription?
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5'-3'
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What is the sigma factor; what is the rho factor
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required for prokaryote initiation and termination of transcription
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What is the product of: RNA polymerase I, II and III
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RNA pol I: 28s, 18s, 5.8s rRNA; RNA pol II: hnRNA/mRNA, snRNA; RNA pol III: tRNA, 5s rRNA
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Steps in prokaryotic production of RNA
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1. Sigma factor + RNA polymerase binds promoter TATA; 2. Transcription 5'-3' begins and sigma factor released; 3. Rho-independent or dependant termination.
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Steps in eukaryotic production of RNA
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1. RNA polymerase II binds promoter region with help of transcription factors. 2. RNA pol II transcribes introns and exons; 3. Ends transcription upon reaching termination sequence.
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Steps of eukaryotic mRNA processing
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1. Addition of 7-methylguanosine cap to the 5' end; 2. Poly-A tail attached to 3' end; 3. Splicing of introns by spliceosomes (snRNA)
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What is a silent mutation?
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New codon codes same amino acid
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What is a missense mutation?
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New codon codes different amino acid
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What is a nonsense mutation?
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New codon codes a stop codon
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What is a frameshift mutation?
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Deletion or addition of a base
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What is a large segment deletion? Mention two examples
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Unequeal crossover in meiosis. Ex. Alpha thalasemia: deletion of alpha globin gene from chromosome 16; Cri-du-chat: 5q deletion
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What is a triplet repeat expansion? Mention an example
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Protein is longer than normal and unstable. Ex. Huntington CAG repeat codes multiple glutamines
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Describe amino acid activation
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1. AA + ATP + tRNA + aminoacyl-tRNA synthetase --> aminoacyl-tRNA + AMP + ppi. AAtRNA synthetase recognizes anticodon sequence of tRNA
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Describe translation by activated aminoacyl-tRNA
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Anticodon sequence of tRNA binds to codon on mRNA (antiparallel and complimentary)
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What is a peptide bond?
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Bond between carboxyl group of one AA to amino group of another AA
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Protein synthesis: describe initiation process
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1. Small subunit of ribosome binds Shine-Dalgarno or 5' cap of mRNA and slides down to first AUG codon; 2. tRNA binds start codon (methionine); 3. Large subunit binds small subunit
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Protein synthesis: describe elongation process
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1. Charged tRNA binds A site and mRNA codon; 2. Peptide bond by peptydyl transferase (uses 2 high energy bonds from aminoacyl-tRNAs); 3. tRNA is removed from P site/growing peptide; 4. ribosome moves 5'-3' exactly one codon. Translocation requires EF-2 (inactivated through ADP-rybosylation by Pseeudomona and diptheria toxins)
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Protein synthesis: describe termination process
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1. Stop codon of mRNA moves to A site; 2. peptydyl transferase releases peptide chain from tRNA in P site; 3. ribosome dissociates
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What are the 4 structures formed during protein folding?
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1. Primary structure: sequence of AA; 2. Secondary: stable a-helix and b-pleaded sheets; 3. Tertiary: interaction between secondary structures and final protein structure; 4. Cuaternary: multiple subunit interaction
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What is the function of ubiquitin?
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Covalently binds to misfolded proteins to signal their destruction by proteasome
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Signal sequence required by proteins destined to be secreted, placed on cell membrane or directed to lysosome
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N-terminal hydrophobic signal sequence
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What is O-linked glycosylation?
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Proteins acquire oligosacchride side chains attached to serine or threonine residues
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What is N-linked glycosylation?
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Proteins acquire oligosacchride side chains attached to asparagine residues
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What does phosphorylation of manose residues do? I-cell disease?
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Manose residues on oligosacchride chain are phosphorylated in golgi aparatus to direct the enzyme to lysosome. In I-cell disease, lysosomal enzymes are secreted into extracellular space due to inappropriate phosphorylation of manose residues
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Mention 4 post-translational covalent modifications
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1. Glycosylation: addition of oligosacchrides; 2. phosphorylation: addition of phosphate by kinases; 3. proteolysis: cleavage and activation of peptide bonds (proinsulin, trypsinogen, prothrombin); 4. y-carboxylation: produces Ca+ binding sites (vitamin K dependant)
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Describe postranslational modifications of collagen
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1. N-terminal hydrophobic signals are added to prepro-alpha chains; 2. hhydrophobic signal is removed in RER; 3. Hydroxylation of prolines and lysines (requires vit C); 4. Glycosylation of hydroxylysines; 5. Triple helical structures self-assemble (procollagen); 6. Secretion of procollagen; 7. Cleavage of propeptides; 8. Cross-linking of collagen by lysil oxidase (requires O2 & Cu+); 9. Aggregation of fibrils into collagen fibers
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Biochemical defect in Scurvy
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Deficient hydroxylation of pre-alpha collagen chains secondary to vitamin C defficiency. Vitamin C is required by lysyl and prolyl hydroxylases
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Biochemical defect in Menkes disease
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Deficient cross-linking of collagen secondary to functional copper deficiency: depigmented hair, arterial tortuosity, osteoporosis, anemia
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Biochemical defect in Osteogenesis imperfecta
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Mutations in collagen genes: skeletal deformities, fractures, blue sclrera
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Biochemical defect in Ehlers-Danlos
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Mutations in collagen genes and lysine hydroxylase gene: hyperextensible fragile skin, hypermobile joints, dislocations
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What is an operon?
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Group of prokaryot genes coding for a group of proteins required for a metabolic function, along with regulatory regions that control gene expression
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The DNA sequence to which activator proteins bind in eukaryotes
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Response elements
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Mention 2 upstream promoter elements
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1. CCAAT boc (-75) that binds transcription factor NF-1; 2. GC-rich sequence that binds SP-1
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The DNA regulatory base sequences in the vicinity of genes
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cis regulators
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What are trans regulators?
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The transcription factors and the genes that code for them
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Mention 4 characteristics of enchancers
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1. Up to 1,000 bases away from gene; 2. Located upstream, downstream or within introns; 3. The orientation is unimportatnt; 4. Tissue specificity if transcription factors are not present in tissue
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What does the activation domain of transcription factors allows them to do?
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Interact with RNA polymerase II to stabilize the formation of the initiation complex
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Mention 3 general transcription factors
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SP-1, NF-1, TFIID (TATA factor)
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Mention 3 specific transcription factors
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steroid receptors, CREB protein PPARs
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Describe the control of gluconeogenesis by response elements
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1. Cortisol + zinc finger receptor bind GRE of PEPCK gene; 2. Glucagon increases cAMP --> protein kinase A --> phosphorylation of CREB --> CREB binds CRE --> upregulation of PEPCK. PEPCK converts OAA into PEP
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What does acetylation of histones do?
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Increases gene expression
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What does methylation of DNA do?
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Silences genes
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What is genetic imprinting?
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Results in mono allelic expression (Prader-Willi, Angelman)
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What does glucose so to lactose operon in prokaryotes?
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Increases intracellular cAMP, which activates CAP (activator protein), which binds CAP site (response element)
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What does lactose do to the lactose operon in prokaryotes?
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Inactivates active repressor and allows b-galactosidase gene transcription
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What are palindromes?
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Sequences of 4-8 bases that are inverted repeats and are recognized by restriction endonucleases. Example: GAATTC
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What should a vector for recombinant DNA contain to be expressed?
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Plasmid with restriction site, resistance to antibiotics gene, promoter and Shrine-Dalgarno sequence
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Genomic DNA Vs. cDNA cloning
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Genomic DNA: cleaved by restriction endonucleases, total DNA is cloned, introns; cDNA: reverse transcription of mRNA, genes expressed cloned, no introns
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Type of material analyzed by Southern blot? Probe?
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DNA. 32P-DNA probe. Used to determine restriction fragments of genes
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Type of material analyzed by Northern blot? Probe?
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RNA. 32P-DNA probe. To measure sizes and amounts of mRNA
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Type of material analyzed by Western blot? Probe?
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Proteins. 125I or antibody. To measure amount of antigen or antibody
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phospholipids of the cell membrane
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phosphatidylcholine, sphingomyelin, phosphatidylentholamine, phosphatidylserine
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flippase
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removes phosphatidylenotholamine and phosphatidylserine from the outer leaflet of the cell membrane using ATP
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glycolipids of the cell membrane
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cerebrosides, gangliosides (contains sialic acid), neutral glycolipids
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NMDA receptor
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require glutamate and glycine to allow Na and Ca influx and K efflux; irreversible antagonists: amantadine and memantine; reversible antagonists: ketamine and PCP
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AMPA receptor
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binds glutamate and allows Ca and Na influx and K efflux; responsible for excitotoxicity
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L-type Ca channel
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also called dyhydropiridine receptor; allows Ca influx and depolarization of cardiac and smooth muscle and interacts with ryanodine sarcoplasmic channels of smooth, cardiac and skeletal muscle
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Fast Ca channel
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also called ryanodine receptor; interacts with the L-type Ca channels to release Ca from the sarcoplasmic reticulum
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T-type Ca channel
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located in the SA and AV nodes (phase 4) and in the thalamus (ethosuximide)
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nACh receptor
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binds ACh and allow Na influx to depolarize muscle; succinylcholine (agonist, depolarizing) and tubocurarine/curonium's (antagonists, nondepolirizing)
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GABAa receptor
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two alpha subunits and 3 beta or gamma; 2 GABA molecules bind the two alpha subunits for influx of Cl
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Gs receptor
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has alpha, beta and gamma subunits; GDP is bound to the alpha subunit; binding of its substrate changes GDP to GTP and units dissociate and adenyl cyclase is activated to increase cAMP; alpha subunit deactivates GTP
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protein kinase A
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phosphorylates seine and threonine residues of intracellular enzymes to increase activity
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zinc finger receptor
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intracellular receptor made up of four cysteines and a zinc atom that has hormone-binding and DNA-binding regions; crosses nuclear membrane to directly influence transcription; TFIIA, Sp1, steroid hormone receptors
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postranslational modifications
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N-linked and O-linked glycosylation, phosphorylation of tyrosine, serine, threonine residues, sulfation, methylation and acetylation of lysine residues, gamma-carboxylation of glutamate, myristoylation of glycine, palmitoylation and fernasylation of cysteine; all happen in the Golgi complex
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clathrin coated vesicles
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for receptor-mediator endocytosis or packaging of proteins destined for secretion and lysosome
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non-clathrin-coated vesicles
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for proteins destined for the membrane
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smooth endoplasmic reticulum
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synthesis of membrane phospholipids, steroids, P450 and glucoronyl transferase enzymes, glycogenolysis via glucose-6-phosphatase, fatty acid elongation, lipolysis via hormone-sensitive lipase, calcium storage
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intermediate filaments
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10-12nm diameter; links extracellular matrix to cytoplasm and nucleus
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microtubules
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25nm diameter; consist of 13 circularly arranged a and b tubulins; associated with dynein (retrograde) and kinesin (anterograde) ATPases; polymerization inhibited by colchicine and vincristine; depolymerization inhibited by paclitaxel
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cytokeratin
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intermediate filament of epithelial cells
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vimentin
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intermediate filaments of endothelial and vascular smooth muscle cells, fibroblasts, chondroblasts and macrophages
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desmin
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intermediate filament of skeletal and non-vascular muscle
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neurofilaments
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intermediate filaments of neurons
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glial fibrillar acidic protein
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intermediate filaments of glia and microglia cells
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cis regulators
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core promoter sequence (TATA, BRE, DPE), proximal promoter region (GC box, CCAAT box), enhancers, silencers, insulators, response elements (CRE, SRE, IRE, GRE, PRE, HSRE, HMRE)
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trans regulators
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general transcription facotrs (TFIIs), gene regulatory proteins, CREB, serum response factor, Stat-1, Mep-1, AP1, steroid hormone receptor
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homedomain proteins
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helix-turn-helix with helix 3 being the DNA binding domain; OCT-1, OCT-2, Pit-1
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leucine zipper proteins
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alpha helix in which every 7th AA is leucine allows dimerization; CREB, Fos, Jun
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helix-loop-helix proteins
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two alpha helixes with a loop in between forms Y-shaped dimer; MyoD, Myc
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transcription initiation complex
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formed by RNA polymerase II and TFIIs at the core promoter TATA
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serum response factor
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trans factor binds SRE in response to serum growth factor
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Stat-1
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trans factor binds IRE in response to IFN-gamma
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Mep-1
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trans factor binds HMRE in response to heavy metals
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AP1
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trans factor binds PRE in response to phorbol esters
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hsp70
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trans binds HSE in response to heat shock
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core promoter region
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TATA interacts with RNA polymerase II and TFIIs
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proximal promoter region
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upstream from the core promoter; CCAAT box and GC-rich sequence
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NFAT
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transcription factor that increases expression of IL-2 gene; when T-cell receptor is enganged --> IP3 --> calcium activation of calcineurin phosphatase --> dephosphorylation of NFAT --> enters nucleus to upregulate transcription
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Pit-1
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gene regulatory homedomain protein required for transcription of GH, TSH and PRL; deficiency leads to pituitary dwarfism and hypopituitarism
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miRNA
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70bp inverted repeat that is cleaved by Dicer to form two interfering RNAs which blocks complimentary sequences of mRNA and prevents expression
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alternative promoter
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the first exon can vary with common downstream exons making different isoforms of same weight
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alternative internal promoter
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transcription is begun in at different promoters within the gene which produces proteins of different weight
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alternative RNA splicing
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spliceosome combines exons in different ways producinf different isoforms of the protein
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lac operon: glucose+ lactose+
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lac repressor is not bound to operator, CAP is not bound due to high cAMP --> lac operon is off
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lac operon: glucose+ lactose-
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lac repressor is bound to operator, CAP is not bound due to high cAMP --> lac operon is off
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lac operon: glucose- lactose-
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lac repressor is bound to operator, CAP is bound to due to low cAMP --> lac operon is off
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lac operon: glucose- lactose+
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lac repressor is not bound to operator, CAP is bound due to low cAMP --> lac operon is on
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satellite DNA
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component of the centromere that binds the mitotic spindle
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