Term
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Definition
| short RNA segment synthesized on the lagging strand during DNA replication and then removed |
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Term
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Definition
| enzyme that joins two adjacent DNA strands together |
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Term
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Definition
| enzyme that opens up the DNA helix |
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Term
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Definition
| newly made strand at the replication fork – made by continuous synthesis in the 5’ to 3’ direction |
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Term
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Definition
| protein complex that encircles the DNA helix and binds to DNA Polymerase |
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Term
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Definition
| enzyme that reversibly breaks phosphodiester bonds and allows the DNA to rotate |
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Term
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Definition
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Term
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Definition
| the point at which DNA is synthesized in the eukaryotic cell cycle |
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Term
| ORC Origin Recognition Complex |
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Definition
| bound to DNA at the origin of replication in eukaryotic chromosomes |
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Term
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Definition
| special DNA sequence where replication begins |
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Term
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Definition
| enzyme that elongates the telomere, a repetitive sequence at the ends of chromosomes |
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Term
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Definition
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Term
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Definition
| insertion or deletion of bases – disrupts the reading frame |
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Term
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Definition
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Term
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Definition
| purine to pyrimidine or vice versa |
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Term
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Definition
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Term
| NHEJ Non-Homologous End Joining |
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Definition
| repairs double strand breaks by linking two ends with little regard for sequence homology |
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Term
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Definition
| collective term for enzyme processes that correct deleterious changes that affect a segment of DNA |
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Term
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Definition
| – genetic exchange between identical (or nearly identical) on two copies of the same chromosome, either sister chromatids or homologous |
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Term
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Definition
| repair process the replaces incorrect nucleotides inserted during DNA replication |
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Term
| NER Nucleotide Excision Repair |
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Definition
| helps fix thymidine dimers formed by UV light |
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Term
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Definition
| repair pathway that involves DNA Glycosylase to recognize altered bases |
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Term
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Definition
| RNA and protein compex that removes introns from pre-mRNA |
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Term
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Definition
| protects mRNA integrity in cytosl – added to 3’ end during processing |
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Term
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Definition
| in prokaryotic initiation – sequence found before the translation start codon |
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Term
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Definition
| the genetic code is degenerate b/c more than one codon specifies the same amino acid |
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Term
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Definition
| important residue at the branch site in intron splicing |
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Term
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Definition
| signal that glucose supply has been exhausted and the cell should use other carbon sources for energy |
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Term
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Definition
| modification of the histone tails provides a code, which imparts information for transcription and chromosome condensation |
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Term
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Definition
| important antibiotics are made against Translation inhibitors |
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Term
| T/F Read in the same direction (5’ to 3’), sequences of nucleotides in the new strand are the same as the parental strand |
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Definition
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Term
| T/F Mismatch can distinguish between parent and progeny because the progeny is hemimethylated |
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Definition
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Term
| T/F Only initial steps in DNA repair are catalyzed by specific enzymes that are unique to that process, and latter steps utilize more general enzymes |
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Definition
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Term
| T/F If the origin of replication is deleted in eukaryotes, the DNA on either side of it is lost |
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Definition
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Term
| T/F Most DNA-protein interactions are done through the major groove |
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Definition
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Term
| T/F The MRN complex recognizes both single stranded and double stranded breaks (F b/c it only recognizes DSBs!) |
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Definition
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Term
| T/F The RNA Polymerase sigma factor recognizes the promoter and forms the open-promoter complex |
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Definition
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Term
| T/F PI3 kinases phosphorylate the hydroxyl groups of all amino acids (F b/c it mostly phosphorylates the S/Ts) |
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Definition
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Term
| T/F During transcription, RNA Polymerase binds downstream of the promoter |
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Definition
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Term
| Double stranded DNA has a higher Tm than another. What properties make this true. |
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Definition
| need to define Tm – the 1st strand shown had 10 C/Gs and the second had 6 C/Gs. The C-G interaction has 3 hydrogen bonds between them and requires more energy to break these bonds. There are only 2 hydrogen bonds between A/T |
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Term
| Describe the difference between nucleosides and nucleotides |
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Definition
-Nucleoside = sugar + base
-Nuclotide = sugar + base + PO4 |
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Term
| Be able to draw all four bases |
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Definition
| -Adenine, Guanine, Cytosine, and Thymine |
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Term
| What are the advantages of cDNA cloning over genomic |
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Definition
| -with a cDNA library, you are able to look at just the gene. A genomic library contains the whole genome(introns, etc)! |
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Term
| Be able to look at a segment of DNA and label it from the 5’-3’ ends and justify why. Also be able to circle the phosphodiester bond |
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Definition
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Term
| Be able to write the amino acid from codons and vice versa |
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Definition
| Open Reading Frames - know the EPA sites of ribosomes and what will be the next coming in |
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Term
| Know how restriction enzymes cut and how many fragments are made |
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Definition
-they cut in double stranded palindromes
blunt or sticky
circular = number of cuts
linear = number of cuts + 1 |
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Term
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Definition
| helps with p53 degradation – expand |
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Term
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Definition
| is a histone variant that serves as a DNA damage marker – C-terminal tail is unique because of the conserved SQ regions that can be phosphorylated |
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Term
| Translesion DNA Polymerase |
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Definition
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Term
| Know the 3 steps in response to a double strand break (elaborate further than this!) |
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Definition
-1. Phosphorylation of special histone (H2ax) (this is a DNA damage marker)
-2. (MRN) bridging complex holding the 2 pieces of DNA together – this stabilizes the fork
-3. Protein kinase (Chk2 & p53) send a signal to repair DNA, stop the cell cycle and/or start apoptosis |
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Term
| Matching of -35 and -10 regions |
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Definition
| -Which has the highest transcription rate? –pick and RNA will bind tightly with this sequence due to good match and have a higher rate of transcription |
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Term
| Two roles of the CTD (C-terminal domain) of RNA Polymerase II |
|
Definition
-1. Gets phosphorylated and releases RNA Pol for elongation from the promoter
-2. Important for processing (5’ cap, Poly A tail, and splicing)
-If she asks for the CTDs importance in processing then you can describe the 2 and 5 position serines |
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Term
Describe:
snRNPs
ribozymes
5' capping |
|
Definition
-snRNPs – made of snRNA and proteins, removes introns from nascent RNA
-ribozymes – catalyzes RNA-RNA interactions during intron lariat removal
-5’ capping – 7 methyl guanosine – protects the 5’ ends of mRNA from exonucleolytic degradation |
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Term
| 2 examples of DNA distortion |
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Definition
-1. cAMP-CAP complex binds
-2. TBP of TFIID binds to the TATA box |
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Term
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Definition
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Term
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Definition
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Term
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Definition
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Term
| KNOW the alpha amanatin levels that correspond!! |
|
Definition
RNA pol I not affected
alpha amanatin inhibits RP II activity at low concentrations (~1 mg/ml)
inhibits RPIII activity at high concentrations (~30 mg/ml) |
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Term
| Know how to do DNA – mRNA strand |
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Definition
mRNA strand . the DNA is 5’ – 3’ , complementary strand 3’– 5’ and mRNA is 5’ – 3’
-from one segment, 3 peptides can be made b/c of reading frames! |
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Term
| Know the picture regarding glucose and lactose levels and whether or not the operon is on or off |
|
Definition
operon turned on when glucose gone (cAMP present)
Lactose present |
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Term
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Definition
| proximity to telomeres (heterochromatin)proximity to telomeres (heterochromatin) |
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Term
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Definition
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Term
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Definition
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Term
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Definition
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Term
|
Definition
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Term
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Definition
Binds directly to transcription activator
Found 1000s of bp from transcription start sites |
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Term
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Definition
| Essential for transcription initiation |
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Term
|
Definition
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Term
|
Definition
|
|
Term
| Eukaryotic origins fire once and only once each cycle (S phase) because |
|
Definition
| Cdc6 is degraded at the start of replication |
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Term
| If DNA helicase travels along the leading strand template into the open replication fork |
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Definition
| it would be a: 3’-5’ helicase |
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Term
| What enzyme is actively involved in proofreading during DNA replication |
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Definition
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|
Term
| General transcription factor TFIID |
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Definition
| is responsible for recognizing the basal promoter elements, such as the TATA box |
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Term
| Alternating sigma factors |
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Definition
| bind to distinct promoter sequences |
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Term
| Addition of ligand switches on a gene by removing the repressor protein. What kind of gene regulator is this? |
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Definition
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Term
|
Definition
| predominantly found in actively transcribed genes |
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Term
| Protein complex that utilizes the energy of ATP hydrolysis to displace/remodel the nucleosomes along DNA called |
|
Definition
| chromatin remodeling complexes |
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Term
| Which possesses histone deacetylation activity |
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Definition
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|
Term
| In eukaryotes, wobble occurs between |
|
Definition
| the 3’ position in the codon and the 5’ position in the anticodon |
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Term
| Translation fidelity is maintained by |
|
Definition
| the amino-acyl and RNAs in the A site have sufficient time to diffuse out of the site if they don't match |
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Term
|
Definition
| responsible for recognizing basal promoter elements, such as the TATA box |
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|
Term
| Alternative sigma factors |
|
Definition
| bind to distinct promoter sequences |
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|
Term
| Addition of ligand switches on a gene by removing the repressor protein. What kind of gene regulation is this? |
|
Definition
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|
Term
|
Definition
| is predominantly found in actively transcribed genes |
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|
Term
| Proetin complexes that utilize the energy of ATP hydrolysis to displace or remodel nucleosomes along a stretch of DNA are called |
|
Definition
| chromatin remodeling complexes |
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|
Term
| Which one of the following protein or complexes possess histone deacetylation activity? |
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Definition
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Term
|
Definition
| a complex of RNA and protein that serves to remove introns from pre mRNA |
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Term
| In order to protect the integrity of an mRNA in the cytosol of a cell |
|
Definition
| a(n) Poly A tail is added to the 3’ end during RNA processing. |
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|
Term
| In prokaryotic initiation, one will see the sequence AGGAGGU just before the translation start codon. This sequence is known as the |
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Definition
|
|
Term
| If more than one codon specifies the same amino acid, the code is said to be |
|
Definition
|
|
Term
| For intron splicing an important residue at the branch site of a nascent mRNA is |
|
Definition
|
|
Term
|
Definition
| is an interacellular signal that glucose supplies have been exhausted and the cell should use alternative carbon source |
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Term
| Some of the important antibiotics are made against |
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Definition
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|
Term
| The modifications of the __________ provide a code that imparts information not just for transcription but for DNA repair and chromatin condensation. |
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Definition
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|
Term
| Phostphates are attached to CTD of RNA polymerase 2 during trascription. What are the various roles of RNA pol 2 CTD phosphorylation? |
|
Definition
1.Phosphorylation of CTD tails is an event that allows release of RNA polymerase from promotor and GTF's.
2. Aids in processing: 5' cap, 3' poly A tails and intron splicing. |
|
|
Term
| Two roles of the CTD (C-terminal domain) of RNA Polymerase II |
|
Definition
-1. Gets phosphorylated and releases RNA Pol for elongation from the promoter
-2. important for processing (5’ cap, Poly A tail, and splicing) |
|
|
Term
| The CTDs importance in processing then you can describe the 2 and 5 position serines |
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Definition
| Two proteins bind to the 2,5 phosphorylated CTD and recognize the termination sequences as they pass by. |
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Term
|
Definition
| made of snRNA and proteins, removes introns from nascent RNA |
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Term
|
Definition
| catalyzes RNA-RNA interactions during intron lariat removal |
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|
Term
|
Definition
| 7 methyl guanosine – protects the 5’ ends of mRNA from exonucleolytic degradation |
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|
Term
| 7 methyl guanosine – protects the 5’ ends of mRNA from exonucleolytic degradation |
|
Definition
-1. cAMP-CAP complex binds
-2. TBP of TFIID binds to the TATA box |
|
|
Term
| Phenominon that causes Ade 2 to give rise to red colonies when it is moved closer to a telomere is _______________. Moving Ade 2 closer to heterochromatin silences genes and causes red colonies. |
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Definition
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|
Term
|
Definition
| sugar + base w/out phoshpate |
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Term
|
Definition
| sugar,base, phosphate bound together by phoshpodiester bond |
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Term
|
Definition
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|
Term
|
Definition
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|
Term
|
Definition
Binds directly to transcription activator
– enhancer
Essential for transcription initiation –
- enhancers and promoters
Recognized by TFIID –
-promoter
Found 1000s of bp from transcription start sites –
-enhancers |
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|
Term
|
Definition
|
|
Term
| Multiple copies of one gene in an organism |
|
Definition
Gene families/
transposons/
paralogs/
LINEs/
SINEs |
|
|
Term
| Two nucleotides in an RNA or DNA molecule that are held together by hydrogen bonds. |
|
Definition
| Complementary pairs, base pairs |
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|
Term
| Complex of DNA, histones, and nonhistone proteins found in the nucleus of a eukaryotic cell. |
|
Definition
| Nucleosome (histone proteins only) to Chromatin to Chromosome (but, at the chromosome stage, the nucleus is degrading/degraded) |
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|
Term
| 4. Describes the polarity of the two strands in a DNA helix; the polarity of one strand is oriented in the opposite direction to that of the other. |
|
Definition
| Antiparallel, right handed helix |
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|
Term
| 2. Dideoxy sequencing uses modified nucleotides as substrate. |
|
Definition
| Dideoxy nucleotides have no –OH group at the 2’ or 3’ of the sugar. They are used to stop DNA replication at a specific base, and to compare those segments against the length of other segments stopped using other dideoxy nucleotides to sequence a DNA segment. However, there are only a few dideoxy nucleotides present in comparison to deoxynucleotides. |
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|
Term
| 3. Genomic DNA library is advantageous compared to cDNA library |
|
Definition
| You can recreate a cDNA library from a genomic library, but you cannot reconstitute a Genomic library from a cDNA library. Since there may be much of interest in introns, better to have all genome available. Also, cDNA library does not contain regulatory DNA sequences. |
|
|
Term
| 4. A small genome size means the organism has less number of genes. |
|
Definition
| If the organism has fewer introns or other types of non-coding material in the genome, they may code for a similar number of genes, but do so in a much more linear and compact way. Example is Fugu fish – similar gene count to other fish/organisms, but very few introns or repeated sequences. |
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|
Term
| 1. Explain what is “semi-conservative” form of DNA replication? |
|
Definition
| When DNA is replicated, the original double strand is split into two template strands. Complementary base pairs are added onto the template strand and each new DNA double helix consists of one of the original strands (template) and its complementary nascent strand. The template strand is conserved (remains intact) and a nascent strand is replicated onto the template strand. |
|
|
Term
| 2. What two activities of DNA polymerase make it a high-fidelity enzyme? |
|
Definition
When complementary base pairs are added to the developing daughter strand, the polymerase creates an environment that has preferential affinity for the correct base pair. The base pair has to undergo a conformational change before being covalently added onto the developing strand, again showing a preference for the correct complementary base pair.
The second error-correction mechanism of DNA polymerase is exonucleolytic proofreading. If an incorrect base pair IS added to the 3’-
OH end of the primer strand, the polymerase clips off any terminal unpaired nucleotides going back until it comes to a correctly paired nucleotide that it can use to continue forward with the correct match. |
|
|
Term
| a) This enzyme helps in joining two DNA strands |
|
Definition
|
|
Term
| b) This protein is important in unwinding DNA helix |
|
Definition
|
|
Term
| c) These proteins bind to single stranded DNA and prevents it from rewinding until replication has occurred |
|
Definition
| Single-strand DNA-binding (SSB) proteins in prokaryotes or RPA (three subunits) in eukaryotes |
|
|
Term
| d) This protein serves as a processivity factor for DNA polymerase |
|
Definition
|
|
Term
| 4. What is deamination and how do cells repair this kind of damage |
|
Definition
Deamination is a spontaneous mutation that typically converts cytosine to uracil, but can occur on other bases as well. It occurs at a rate of about 100 bases per cell per day. If left without repair, this could cause base pair changes after replication as the uracil would cause an adenosine to pair with it on the nascent strand, causing frequent and potentially devastating point mutations.
Deaminated Cs or As are typically repaired with enzymes called DNA glycosylases via the base excision repair pathway. Different glycosylases recognize specific types of altered bases and catalyze its hydrolytic removal. The missing base pair gap is recognized by an enzyme called AP endonuclease which cuts the DNA backbone, after which the damaged base is removed and the gap is repaired. DNA polymerase adds the correct nucleotide and DNA ligase completes the nick. |
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|
Term
| 1. Briefly explain how lac operon combines both positive and negative controls |
|
Definition
The lac operon is responsible for 3 genes that allow for the intake/transport and cleavage of lactose. However, the cell does not require these proteins unless lactose is present and other preferable energy sources are not present. Positive control is maintained through the CAP protein. When glucose levels are low in the cell, cAMP is formed. cAMP then binds CAP and activates transcription of lac Z. The lactose repressor is bound to the DNA across the promoter site while lactose levels are low. When lactose levels rise, the lactose inhibits the lactose repressor and it dissociates with the lac promoter, allowing transcription to occur.
For transcription to occur, both cap/cAMP must be bound and active (glucose levels low) and lac repressor/lactose must be bound to remove the repressor (lactose levels high). |
|
|
Term
| 2. What is catabolite repression |
|
Definition
| Catabolite repression occurs in bacteria when glucose, a preferable source of Carbon, is present. It prevents expression of alternative pathways for Carbon sources. When glucose is not present, cAMP is produced which binds and activates a promoter, CAP, which then promotes transcription of genes used in alternate carbon source formation (lac operon promotion). |
|
|
Term
| 3. What are the different types of eukaryotic RNA polymerases and how do they differ in the genes transcribed |
|
Definition
| RNA polymerase I transcribes pre-rRNA proteins (5.8S, 18S, and 28S rRNAs). RNA polymerase II transcribes Pre-MRNA (primarily, non-rRNA) and some snRNA. RNA polymerase III transcribes an additional rRNA protein (5S), some small RNAs, and some additional snRNAs. |
|
|
Term
| 4. Give two examples where DNA is subjected to distortion to carry out normal cell functions |
|
Definition
DNA distortion is found where TBP (TATA Binding Protein) binds the minor groove of DNA and creates a complex with DNA at the TATA Box-containing fragment. This causes a bend in the DNA.
DNA is looped when Transcriptional activators/enhancers are artificially linked to a promoter from a different gene. This allows the DNA between the enhancer and the promoter site to loop during transcription |
|
|
Term
| 1. What are heat shock proteins? How do Hsp60 proteins differ from Hsp70 proteins? |
|
Definition
Heat Shock Proteins (Hsp), are types of chaperone proteins that help other proteins fold. When the temperature increases, more Hsp are made to help refold denatured proteins. In general, Hsp bind to hydrophobic patches and require ATP hydrolysis.
Hsp70 bind proteins as they exit the ribosome and give order/specificity to how hydrophobic regions interact in the cytosol and gain a specific interior arrangement.
Hsp60 form a basket-like conformation creating a hydrophobic chamber and bind already synthesized proteins, giving them time to refold in a specific manner without interactions with other cellular proteins. |
|
|
Term
| 2. What are the functions of the different subunits of the proteasome? |
|
Definition
| A proteasome is made up of two subunits, a 19S regulatory particle, and a 20S core particle. The regulatory subunits of proteasome have many functions, including specific substrate recognition of the polyubuitin and protein, ubiquitin releasing using deubiquitinizing enzymes that hydrolyze isopeptide bonds in three steps (for recycling of the ubiquitins), and substrate unfolding using hexameric unfoldase. This allows the protein to enter the core particle prepared for its protease activity. The core consists of separate proteases bound together. Different types of protease activities are built into the core: Trypsin-like activity, chymotrypsin-like activity, and post-glutamyl-peptide hydrolytic activity. |
|
|
Term
| 3. Describe the process of ubiquitination. |
|
Definition
| Ubiquitination is a process of attaching ubiquitin (a protein of 76 amino acids) covalently to target proteins by an iso-peptide bond made from the C-terminal glycine of ubiquitin binding the epsilon-amino of lysine residues on the target protein, and then attaching additional ubiquitins (polyubiquitination) between the C-terminal glycine of one ubiquitin to the amino of lysine 48 on a second ubiquitin. The isopeptide bond is made in three steps utilizing three additional proteins, E1, E2, and E3. E1, the ubiquitin activating enzymes loads a ubiquitin to itself through a thioester linkage with the C-terminal glycine of a cysteine side chain. The ubiquiting conjugation protein, E2, transfers the ubiquitin from E1 as a sulfur ester onto another cysteine on E2 , and E3, ubiquitin ligase, transfers the ubiquitin to the target protein using very target-specific E3s. |
|
|
Term
| 4. How do epigenetic changes lead to development of cancer? |
|
Definition
| Epigentic changes alter gene expression by creating a more euchromatic or heterchromatic region via methylation and/or the histone code, changing the amount of transcription that area of DNA (and those genes) will receive. If an area becomes more densely packaged via methylation, or changes in histone makeup, the genes involved will be turned off. If the area is methylated more, or gets a different histone makeup, it may cause further transcription of the related genes. |
|
|
Term
| 5. Write three major differences between “Necrosis” and “Apoptosis”. |
|
Definition
Since apoptosis is a programmed death following a specific pathway, and necrosis is and accidental death, there are differences in how the cell reacts.
Apoptosis is signal induced, affects the single cell receiving the signal, and goes into an orderly and predictable path of cell shrinkage, cytoskeleton collapse and membrane blebbing, release of mitochondrial proaptotic proteins (cytochrome C), nuclear lamina disruption, chromatin condensation and orderly fragmentation, and formation of apoptotic bodies that are ingested by neighboring cells. And since there is no release of inflammatory chemicals, there is no inflammatory response or macrophage recruitment.
Necrosis can cause cell swelling and loss of membrane integrits, spilling out its contents. This can be caused by an acute insult which may affect other neighboring cells as well. The cytosolic release of organelle contents, lysomic reactants, and random DNa fragments causes a significant inflammatory response which signal for macrophage phagocytosis. |
|
|
Term
| Morphological changes in apoptotic cells |
|
Definition
Cells shrink and condense
The cytoskeleton collapses
Nuclear envelope disassembles
Nuclear chromatin condenses and breaks up into fragments.
Cell surface breaks up into membrane enclosed fragments called apoptotic bodies.
These apoptotic bodies are then engulfed by neighboring cells or macrophages. |
|
|
Term
| apoptosis nuclear condensing |
|
Definition
|
|
Term
| apoptosis nucleus fragmenting |
|
Definition
|
|
Term
|
Definition
Apoptosis is programmed cell death
Necrosis (accidental cell death
-Cells swell and burst spilling their content |
|
|
Term
| Apoptotic cells are biochemically recognizable |
|
Definition
During apoptosis endonucleases cleaves chromosomal DNA in linker regions between nucleosomes
Creates fragment of distinct sizes when run on gel electrophoresis
2. TUNEL assay (TdT-mediated dUTP Nick End Labeling)
Cleavage of DNA generates new DNA ends, which can be detected by TUNEL techniques which labels the ends of DNA fragments in the nuclei of apoptotic cells.
The enzyme terminal deoxynucleotidyl transferase (TdT) adds chains of labeled dUTP to the 3’-OH ends of DNA fragment.
3. Detection of Annexin V
Due to changes in the plasma membrane during apoptosis, the phospholipid phosphatidylserine flips from the inner to the outer leaflet of the lipid bilayer and gets exposed on the cell surface.
Annexin V binds phosphatidylserine and can be labeled for detection.
•Aside from being a marker, annexin V serves two purposes:
1) it serves as an “eat me” signal to macrophages to ingest (phagocytose) apoptotic cells
2) it blocks the inflammation associated with phagocytosis |
|
|
Term
| Apoptosis is activated through two major signaling pathways |
|
Definition
Intrinsic pathway
Intrinsic pathway is triggered
from within the cell by stress or
DNA damage
Extrinsic pathway
Extrinsic pathway is activated
when a pro-apoptotic ligand binds to a pro-apoptotic receptor
Both lead to caspase cascasde |
|
|
Term
| Caspases are intracellular enzymes that trigger cell death |
|
Definition
Caspases are proteases that have a cysteine at their active site and
cleave their target proteins at specific aspartic acids.
Inactive precursor molecule is procaspase which when
proteolytically cleaved become active. |
|
|
Term
| Intracellular Proteolytic Cascade |
|
Definition
Once active, caspases cleave and activate other procaspases thus amplifying proteolytic cascade.
Procaspases which operate in start of proteolytic cascades are called initiator caspases
When activated, initiator caspases cleave and activate executioner caspases which cleaves target proteins. |
|
|
Term
| Cell-surface death receptors activate the extrinsic pathway of apoptosis |
|
Definition
Extracellular signals bind to death receptors on cell surface and trigger extrinsic pathway.
These receptors are transmembrane proteins that contain a death domain which is required for receptors to activate apoptotic program.
These receptors belong to TNF (Tumor necrosis factor) family of receptors. |
|
|
Term
|
Definition
Fas-associated death domain
Death-inducing signaling complex |
|
|
Term
| Intrinsic pathway of Apoptosis depends on mitochondria |
|
Definition
Intrinsic apoptosis depends on the release of mitochondrial proteins in the cytosol where they activate a caspase proteolytic cascade that leads to apoptosis.
Cytochrome c - a water soluble component of electron
transport chain in Mitochondria. When released into cytosol, it binds to procaspase activating adaptor protein called Apaf1. This binding causes Apaf1 to oligomerize to
form wheel like heptamer called apoptosome.
Intrinsic apoptosis depends on the release of mitochondrial proteins in the cytosol where they activate a caspase proteolytic cascade that leads to apoptosis.
Apaf1 in apoptosome recruits initiator procaspase 9 which in turn activates downstream executioner procaspases to induce apoptosis |
|
|
Term
| Intrinsic pathway of apoptosis is regulated by Bcl2 family of proteins |
|
Definition
| Bcl2 family of proteins work mainly by controlling the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol. |
|
|
Term
|
Definition
anti-apoptotic
In the absence of an apoptotic signal, active anti-apoptotic Bcl2 proteins bind to pro-apoptotic BH123 proteins inhibiting oligomerization and activation of apopotosis |
|
|
Term
|
Definition
pro-apoptotic
Pro-apoptotic BH123 oligomerization causes the release of cytochrome c
Makes a pore in mitichondrial membrane |
|
|
Term
|
Definition
pro-apoptotic (PUMA)
Pro-apoptotic BH3-only proteins promote apoptosis by inhibiting anti-apoptotic Bcl2 proteins |
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Term
| Inhibitors of apoptosis (IAPs) bind and inactivate caspases |
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Definition
| anti-IAPs released from BH123 block IAP process and allow caspase cascade |
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Term
| Extracellular survival factors inhibit apoptosis |
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Definition
| Limited amounts of survival factor is released allowing survival of small population of nerve cells, allowing the survival of only best-connected neurons; the remainder undergo apopotosis |
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Term
| Inefficient apoptosis leads to disease |
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Definition
Autoimmune disease
Anti-self lymphocytes are not all killed (using the Fas pathway) as they should be in the thymus or spleen.
Tumors and cancer
Cells with badly damaged DNA are not killed as they should be, leaving them to accumulate more mutations, some of which will make the cancer cells more malignant. |
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Term
| Two properties of cancer cells |
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Definition
They reproduce in defiance of normal constraints on cell growth.
They invade and colonize territories normally reserved for other cells |
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Term
A ___________________ glandular tumor remains inside the basal lamina that marks the boundary of the normal structure
A _________________ glandular tumor destroys duct integrity and spreads to other tissues |
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Definition
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Term
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Definition
| secondary tumors derived from cells that break off of the primary tumor – make cancer hard to eradicate. |
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Term
| Cancer Classification (based on tissue and cells from which they arise): |
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Definition
Carcinomas – derive from epithelial cells (e.g. breast, digestive organs).
Sarcomas – derive from connective tissue or muscle cells.
Leukemias and lymphomas – derive from white blood cells |
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Term
| Mutations underlie cancer development and progression. _________ (production of cancer) often proceeds from mutagenesis (mutation in DNA sequence): |
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Definition
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Term
| A single mutation is not enough to cause cancer |
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Definition
Cancer typically requires more than one mutation in a given cell to trigger uncontrolled growth.
The incidence of cancer rises steeply as a function of age, presumably because somatic cells take additional time to acquire mutations.
Cancer is caused by a progressive accumulation of random mutations.
The incidence of cancer rises steeply as a function of age, presumably because somatic cells take additional time to acquire mutations.
Cancers develop gradually from increasingly aberrant cells
Incidence of cancer increases upon exposure to a carcinogen, as indicated here for bladder cancer among chemical workers exposed to a carcinogen |
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Term
| A small population of cancer stem cells maintain tumors |
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Definition
| Cancer stem cells are capable of indefinite self-renewal, but they also produce a large number of transit amplifying cells with limited capacity for self-renewal. As a consequence, most cells in a tumor are not capable of independently forming tumors. |
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Term
| How do cancer stem cells arise |
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Definition
Cancer stem cells can arise from a normal tissue stem cell that acquires mutations leading to the cancerous state. Alternatively, a highly differentiated cell (transit amplifying cell) can acquire the capacity for prolonged self-renewal.
Cancer is so difficult to eradicate because all cancer stem cells must be killed in a patient. |
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Term
| To metastasize, malignant cancer cells must survive and proliferate in a foreign environment |
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Definition
1. Cancer cell should break free from its neighboring environment.
2. Should be able to survive and proliferate in a new environment |
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Term
| Tumors induce _______________ |
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Definition
angiogenesis
Tumors induce angiogenesis or the development of new blood vessels that feed the tumor. As the tumor grows, it becomes hypoxic and this initiates a signaling pathway that directs the formation of new blood vessels. The vessels not only help supply the tumor with nutrients and oxygen, but also provide an escape route for its cells to metastasize. |
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Term
| Cancer cells do not act in isolation but are supported by the ________ – epithelial cells such as fibroblasts and white blood cells that help sustain the tumor. |
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Definition
stroma
Cancer treatments could be directed against the activated stromal cells, in addition to the tumor cells themselves |
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Term
Hundreds of genes have been found to be altered in various human cancers. They are collectively referred to as cancer-critical genes
Two broad classes of cancer-critical genes are |
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Definition
Proto-oncogenes: overactive or overexpressed forms of these genes promote cancer and are called oncogenes
Tumor suppressor genes: inactive forms of these genes contribute to cancer development. Need both genes inactivated (2 mutations)
DNA maintenance genes could be considered a third class of cancer-critical genes. Mutations in these genes promote genome instability, often activating proto-oncogenes or inactivating tumor suppressor genes. |
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Term
| Proto-oncogene activation (4) |
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Definition
-Deletion or point mutation in coding sequence
-Regulatory mutation
-gene amplification
-chromosome rearrangement |
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Term
| Finding tumor suppressor genes |
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Definition
| Characterization of hereditary retinoblastoma led to the discovery of tumor suppressor genes. The Rb gene encodes a regulator of the cell cycle. In its absence, cell division is uncontrolled. Tumor arises when both copies of the gene are lost or inactivated |
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Term
| The hunt for critical cancer genes continues |
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Definition
| In this approach, DNA from tumor cells and normal tissue are labeled with red and green fluorescent tags, respectively. The DNA is then hybridized to a DNA microarray (DNA chip) in which each spot corresponds to a defined region of the genome. The ratio of red to green fluorescence identifies regions of gene amplification and loss that have occurred in the tumor cell line. |
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Term
| Genetically engineered mice have helped in the study of cancer-critical genes |
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Definition
In this experiment, the incidence of tumors are plotted for three transgenic mice that overexpress various oncogenes.
Single oncogene is not sufficient to turn a normal cell into a cancer cell. |
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Term
| The _______ _________ protein inhibits entry into the cell cycle by repressing S-phase genes |
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Definition
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Term
| The pathway by which Rb controls cell cycle entry contains both proto-oncogenes and tumor suppressor genes |
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Definition
Green: proto-oncogenes
Red: tumor-suppressor genes
E2F is in blue because it can act as either depending on its interacting partners.
Some Glioblastomas & breast cancers – amplified Cdk4 and cyclin D
Deletion or inactivation of p16 is common in many human cancers. |
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Term
Phosphoinositide 3-kinase (PI 3-kinase)/
Akt intracellular signaling pathway is critical for controlling cell growth |
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Definition
Cell growth is regulated by extracellular factors, which bind to receptors on the cell surface and activate intracellular signaling pathways that stimulate accumulation of proteins and other macromolecules.
One of the main signaling pathways activated by growth factors is PI3 kinases, which activates TOR pathway. TOR activates other genes that stimulate protein synthesis and inhibit protein degradation.
Akt drives cell growth by
Stimulating glucose uptake
Increased glucose utilization
Conversion of excess citric acid into acetyl CoA
Net Result
1. Increased lipid synthesis
2. Increased protein synthesis
3. New membrane production |
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Term
| PTEN is the tumor suppressor gene |
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Definition
Loss of PTEN phosphatase is common in many different cancers.
PTEN phosphatase dephosphorylates the molecules that PI3 kinase phosphorylates.
In total, it limits Akt activation and hence controls cell proliferation. |
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Term
| Modes of action of the p53 tumor suppressor |
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Definition
p53 provides protection that does not allow activated oncogenes such as Ras or Myc to cause tumors.
However, mutations in the p53 gene allow many cancer cells to survive and proliferate despite DNA damage. |
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Term
DNA damage activates p53 and blocks
cell division |
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Definition
Cells have checkpoint proteins (such as p53) that can detect DNA damage and prevent progression through cell cycle.
DNA damage can be caused by radiation or other chemicals.
Cell cycle can be arrested in late G1 or at G2/M checkpoint.
The p53 protein is a transcription factor that regulates the transcription of many genes including p21.
The p21 gene encodes a protein p21, that inhibits G1/S and S-Cdk.
This stalls the cell cycle and even leads to cell death unless the DNA damage is repaired.
DNA damage activates p53 and blocks
cell division |
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Term
| p53 induces apoptosis by inactivating anti-apoptotic Bcl2 protein |
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Definition
| Cell defective in p53 fail to show p53-dependent stress responses. They tend to escape apoptosis and if their DNA is damaged, they result in chromosomal abnormalities |
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Term
| Replication of damaged DNA leads to chromosomal abnormalities |
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Definition
Chromosomal abnormalities such as activation of oncogenes or loss of tumor suppressor genes
breakage-fusion-bridge cycle |
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Term
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Definition
Cells with DNA damage to continue through cell cycle.
It allows cells with damaged DNA to escape apoptosis.
Cells with damaged chromosomes lead to GENETIC INSTABILITY which is a hallmark of cancer cells.
Cells become resistant to anticancer drugs and irradiations. |
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Term
Regulation
-Genome level
-DNA level
-RNA level
-Protein level |
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Definition
Access to DNA (chromatin status)
Promoter status
Availability of tsx factors
Activity of tsx factors
mRNA stability
mRNA processing
Post-translational modification
Protein stability |
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Term
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Definition
RNA interference is a
PATHWAY
whereby the expression of specific gene/s are interfered with (inhibited).
RNAi (RNA interference):
A system by which short RNA oligonucleotides (20-25 nts) in a protein complex regulate gene expression
RNA – TARGETS PROTEIN COMPLEX TO THE GENE
Protein Complex – INHIBITS GENE EXPRESSION |
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Term
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Definition
The RNA component (20-25nts) is a short single strand of RNA that provides specificity.
The protein component is made up of a collection of nucleic acid binding proteins, nucleases, and other enzymes that interact with RNA to regulate gene activity. |
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Term
| Where does the RNA come from? |
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Definition
Exogenous source – infection by RNA viruses
siRNA: short-interfering RNA, 21-25 nt.
Mostly exogenous origin.
dsRNA precursors
May be target specific
Degrade the target RNA
Endogenous source
miRNA: microRNA, 21-25 nt.
Encoded by endogenous genes. Humans have about 700miRNAs. Expressed during development. These miRNAs regulate between 30-60% of the 20,000 protein coding genes (between 6,000 and 12,000 genes)
Hairpin precursors
Recognize multiple targets.
Can trigger transcriptional inhibition or mRNA cleavage |
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Term
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Definition
Initiation
Generation of mature siRNA or miRNA
Execution
Silencing of target gene
Degradation or inhibition of translation |
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Term
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Definition
Cropping
Dicing
Activation
Primary-miRNA (pri-miRNA)
-cropping
Precursor-miRNA (pre-miRNA)
-dicing
dsRNA
-activation
ssRNA + protein |
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Term
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Definition
Pasha – dsRNA binding protein
Drosha – RNase III (cleaves dsRNA
Step 1: Cropping (occurs in nucleus)
The microprocessor complex made up of Pasha and Drosha “crop” pri-miRNA to form pre-miRNA, which is exported to the cytoplasm. |
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Term
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Definition
Dicer – RNase III
Step 2: Dicing (occurs in Cytoplasm)
Dicer binds and “dices” pre-miRNA to form short dsRNAs.
Dicer has a “ruler” built into its structure that allows it to “dice” the pre-miRNAs into short dsRNAs of the same length.
Dicer cleaves liberating a ~22 nucleotide RNA duplex with 2 bp 3’overhangs.
Dicer – How does it make those small RNAs so consistently?
Dicer has a built-in “ruler” |
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Term
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Definition
Activation is the process of combining the short miRNA with effector proteins to form a functional complex
The activated RNAi pathway generates mature RNA-induced silencing complexes (RISCs)
Argonaute proteins are key members of RISCs
Humans have 4 Argonaute proteins
C. elegans have 29
Activation is the process of combining the short miRNA with effector proteins to form a functional complex
The activated RNAi pathway generates mature RNA-induced silencing complexes (RISCs)
RISC Formation
dsRNA generated by dicer is bound by RISC-loading proteins
Once loaded, Argonaute (Ago2) “selects” ONE strand of the dsRNA
This is the GUIDE STRAND
The opposite strand (PASSENGER STRAND) is either degraded or otherwise removed
Following Dicing, dsRNA is incorporated into RISCs
Argonaute proteins are critical components of RISCs
Only one of the two strands is incorporated into the final holo-RISC
Guide strand – retained in holo-RISC
Passenger strand – discarded/degraded |
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Term
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Definition
How do the miRNAs know which genes to target?
The miRNA (guide strand) has a seed sequence at the 5’ end that is complementary to the target mRNA. |
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Term
| What does RNAi actually do (EXECUTION)? |
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Definition
Alters DNA itself & blocks Transcription
Heterochromatin formation
Modifies histones (lysine methylation)
Blocks translation
Trashing mRNA
Mess up of the translation machinery |
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Term
| Translation inhibition: To cleave or not to cleave? |
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Definition
If the miRNA is complementary to the target – Argonaute cleaves the mRNA.
Incomplete complementarity – translation of the mRNA is inhibited |
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Term
| RNAi pathways regulate viral gene expression |
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Definition
HCV (Hepatitis C virus) infects liver cells where it causes cirrhosis and sometimes cancer.
HCV infects human liver cells that express an endogenous miRNA (miR-122).
HCV cannot replicate in cells that do not express miR-122.
So by blocking miR-122 expression, we can repress viral infection.
This has been done in HCV-infected Chimpanzees.
FUTURE ANTIVIRAL THERAPUETICS |
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Term
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Definition
miRNA
-Mixed (not perfect) complementarity to target mRNA
-Can trigger transcriptional inhibition or mRNA cleavage
-Tsl inhibition is more common than cleavage
-Endogenously presented.
-Humans have >700 miRNAs.
siRNA
-Perfect complementarity to target mRNA
-Generally cleaves and degrades mRNA.
-Exogenous such as viruses
-Synthetically produced to be used for experiments |
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Term
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Definition
| A set of proteins that help cellular proteins fold correctly or refold correctly |
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Term
| Hsp (heat shock proteins) |
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Definition
Chaperones are heat shock proteins, i.e., made in large quantities at high temperature. Proteins are more likely to “denature” at high temperatures and require refolding.
These proteins have two properties: 1) they bind to hydrophobic patches and 2) require ATP hydrolysis |
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Term
| Two types of heat shock proteins |
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Definition
1. hsp70
bind to exposed hydrophobic amino acid sequences as polypeptides exit the ribosome
- appear to prevent hydrophobic regions from aggregating in a nonspecific manner
hsp60
Acts after a protein has been synthesized
forms basket with hydrophobic sides
- confinement gives protein time to refold without interference of other cellular proteins |
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Term
| The proteasome consists of a core capped by two regulatory particles |
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Definition
19S regulatory particle
20S core particle
19S regulatory particle
Some of the subunits of the core are proteases that hydrolyze peptide bonds.
Core is stack of four heptameric rings
In the regulatory particle base, hexameric unfoldase |
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Term
| Some proteases have specificity. |
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Definition
Digestive proteases:
trypsin — carboxyl side of lysine and arginine (positively-charged residues), except when followed by proline)
- chymotrypsin — carboxyl side of tyrosine, tryptophan and phenylalanine (all contain aromatic rings) |
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Term
| Three subunits of the beta rings are proteases |
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Definition
1. trypsin-like activity
2. chymotrypsin-like activity
3. post-glutamyl-peptide hydrolytic activity (cleaves after acidic or branched-chain amino acid) |
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Term
| The regulatory subunits have many functions |
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Definition
functions:
1. substrate recognition
2. ubiquitin releasing
3. substrate unfolding |
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Term
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Definition
The unfoldase subunits are AAA proteins that hydrolyze ATP as they work. They are related to hexameric DNA helicases.
The unfoldase is normally processive, working residue by residue to unfold the protein.
(AAA ATPase Associated with diverse cellular Activities) |
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Term
| The recognition signal is polyubiquitin |
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Definition
| Ubiquitin is a protein of 76 amino acids, highly conserved among eurkaryotes. Discovered in early 1980s and the discovery won Nobel Prize in 2004. |
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Term
| Ubiquitin is covalently attached to target proteins |
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Definition
| The isopeptide bond is made from the C-terminal glycine of ubiquitin to the epsilon amino of lysine resides on target proteins |
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Term
| Polyubiquitin is made by adding ubiquitin to ubiquitin |
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Definition
| Polyubiquitin chains are made between the C-terminal glycine of one ubiquitin to the amino of lysine 48 on a second ubiquitin |
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Term
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Definition
| The regulatory complex contains one or more enzymes, dubbed DUBs (deubiquitinating enzyme), that hydrolyze the isopeptide bond to recycle ubiquitin monomers. |
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Term
| The isopeptide bond is made in a series of three steps. |
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Definition
E1 — ubiquitin activating enzyme (high-energy thioester linkage to a cysteine side chain on E1)
E2 — ubiquitin conjugating protein (activated ubiquitin is transferred to cysteine on E2)
E3 — ubiquitin ligase
E3, ubiquitin ligase, is responsible for recognizing the target protein
Ubiquitin ligase transfers ubiquitin to the target protein and also builds polyubiquitin chains. |
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Term
| The E3 ligases constitute more than one gene family |
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Definition
| Note that RING E3s transfer ubiquitin to the target directly from the E2, while HECT E3s themselves are ubiquitinylated before transfer. |
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Term
| The ubiquitin conjugation system is used for more purposes than protein degradation |
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Definition
Some target proteins are monoubiquitylated (not polyubiquitylated). This may serve to target proteins to cellular locations.
Cells possess other ubiquitin-like proteins that are usually added singly to targets. SUMO —small ubiquitin-like modifier is one such paralog that aids in controlling the activity of target proteins.
3. Polyubiquitin chains can form between Lys6, Lys11, Lys27, Lys29, Lys33 and Lys63, not just Lys48. The meaning of these alternative linkages is not currently known. |
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Term
| To add complexity, ubiquitin signals other functions besides protein degradation |
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Definition
MONO
histone regulation
MULTI
endocytosis
POLY
proteasomal degradation
DNA repair |
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Term
| A number of human diseases are caused by proteins that misfold and wreck havoc before they can be degraded |
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Definition
Stack of b sheets.
Resistant to proteolysis
These aggregates form amyloid plaques which are prominent in Huntington’s disease and Alzheimer’s |
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