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143 Cards in this Set
- Front
- Back
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What is the nuclear pore complex? |
8 subunits which makes up a pore. The part are FG-repeat domains of FG nuclepporins, transmembrane ring, central scaffold, cytoplasmic ring, nuclear envelope, central channel, nuclear ring, and nuclear basket.
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What is the nuclear matrix? |
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What is chromatin nuclear enveleope> |
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What are nucleoporins? |
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What is the nuclear localization signal? |
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What are importins? |
Soluble heterodimers which bind to NLS-containing proteins
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What are histones? |
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What are nucleosomes? |
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What is the scaffold? |
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What are topoisomerases? |
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What is in the anatomy of a nucleus? |
A nucleolus, nuclear matrix, chromatin fiber, nuclear pores, and the nuclear envelope (double membrane) |
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What is chromatin? |
DNA/protein complex that constitutes chromosomes. Approximately equal amount DNA/protein. Proteins are histones and proteins involved in DNA metabolism. |
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What proteins connect the cytoskeleton to the interior of the nucleus? |
Nesprin protiens. |
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Inside the nuclear membrane, what forms the sheet which attaches to the Nesprin? |
Lamina |
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Which nesprin attaches to Actin? |
Nesprin 1 and 2 |
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What does Lamina look like under a mciroscope? |
A mesh, like a shirt |
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What is Lamina? |
Layers of proteins which overlap and create a mesh as protection. |
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How many subunits makes up the nuclear pore? |
8 subunits, 30-50 proteins each |
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In the NPC, what is the FG-repeat domain of FG nucleoporins? |
F stands for phenylaline, G for glycine, and there is an area which has these repeated. It is an unordered array of fibers in the pore, and it is hydrophobic. |
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RNA and RNA protein complexes move out, and what goes in? |
Proteins move in such as enzymes associated with DNA replication. |
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How do proteins get through the pore? |
SMall proteins flow through, and large ones possess a nuclear localization signal (one to several different regions of a protein that contain short streches of positively charged amino acids). NLS interacts with receptors which assist in transport. |
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How do large proteins enter? |
The protein interacts with importin alpha, and beta, and they interact with a cytoplasmic filament on the pore, and this induced a structural change. The transported molceule goes through the pore. |
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How is importin separated from the protein it carried> |
Ran GTP as an affinity for importin beta, and it binds to beta, which makes the importins and the protien all let go of each other. |
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How are proteins exported through the pore?
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Exportin then guides crries the protein into the cytoplasm. The RanGTP becomes RanGDP, and is released from the exportin. |
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What is chromosome packaging? |
The pasta analopgy: decondensed allows access for enzymes of DNA metabolism (spaghetti), and condensed permits segregation without tangling (breadsticks). This is done by histones. |
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What amino acids are histones made of? |
About 20% of each histones is made of either Arginine or Lysine. |
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What are core histones? |
H2A, H2B, H3, H4, same in most animals |
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What is histone H1? |
A linker histones, much less conserved. Different from mice etc. Unique to each animal. |
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What are the interactions of histones and DNA? |
Compaction involves ionic bonds between - charged phosphates of DNA and the + LYS and ARG of histones. Then hydrogen bonds between DNA backbone and amino acids are formed. Then non-covalent interaction between histones. |
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What is a histone octamer? |
Basic unit of DNA packaging. Made of two copies of each of the four core histones. Make a ball, and DNA is wrapped twice around the ball. H1 associates where the DNA enters and exits nucleosome. |
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What is the beads on a string formation? |
The beads are the nucleosome, and the links are H1 connecting the DNA together. |
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What happens if you add H1 to the histone complex? |
It makes a solenoid, a coled coil. Turns the 10nm fiber into 30nm. |
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What is the heirarchy of chromatin packaging? |
It goes from DNA double helix, nucleosome core particle, nucleosome filament, 30 nm fiber, protein scaffold of a looped domains, to metaphase chromosome. |
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What anchors the loops of protein to the scaffold? |
Cohesin anchors it to the scaffold |
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In |
In the nucleus, just after replication, they remain in loops still because of all the conhesin |
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How are supercoils formed? |
Inbwteen interphase and prophase, condensin comes in and super coils the chromatin. |
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How do you disentangle the chromatin? |
Topsisomerase does this. Type 1 alters superhelical density, and unwinds the strands. This forms a super coil, this occurs in transcription. It first binds and cuts one of the strands of the double helix. This gets rid of all the tension, and a phosphodiester bond reseals nick. |
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What is type II topoisomerase? |
A detangling enzyme, the magicians rings. You have two chromatin fibers, one called G (gate) segment, and it opens up to let in transport segment (other chromatin). With 2 ATP, it chages confirmation and then this cuts the G segment, and the enzyme binds to the two ends, and then it changes confirmation again, so that the T segment goes through, and then it puts the two ends together again. |
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What are loop domains? |
So if there is an open loop which the cell doesn't need at the moment, then chromatin regulator comes in compacts that loop. It becomes heterochromatin, and silent (it cannot be read). And insulator elements prevent the surrounding loops from compacting. |
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What is FISH? |
Fluroescense in situ hybridization, and false coloured images are merged so you can see that certain chromosomes are located at certain areas in the cell. |
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What happens if you induce genes TTF and GREF by estrogen? |
Within 60 min the genes on separate chromosomes far away move towards each other and colocalize. This shows that chromosomes move. |
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What are Transcription factories? |
There are places in the nucleus where transcirptional machinery is localized and the genes go to those factories to be transcribed. There are 30-50 sites. |
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What is a regulating gene? |
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What isa represser? |
etxra
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What is an inducer? |
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What is co-repressor? |
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What is a derepression? |
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What is positive vs negative control? |
Depends on the active form of the trans-acting factor (repressor), and its effect upon binding to its target cis-acting sequence. Do they protote transcription? or do they repress it? |
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What is cis vs trans acting factors? |
Cis-acting promotor sequence to which a trans-acting transcription factor binds. Cis means "on the same strand", and that means the binding site for the trans (transcription factor), is on the same strand as The trans binds to the promotor site and this is on teh same region of the gene as the gene it controls. |
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WHat are deletion mutants? |
extra
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What is an enhancer? |
DNA elements foten located far away from the gene they control. Orientation independent. |
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Explain lactose metabloism in e.coli. |
Occurs in the absense of glucose. Lactose is galaactose and glucose. Lactose is the inducer, and induction takes place. So the bacteria turns on the lac-operon. This encodes the enzyme beta-galactosidase, and it looks for lactose to express the glucose. At around 3 min add lactose, then mRNA ramps up for 10 min, then beta-galactosidase comes around 2 min after mRNA, and then lasts. |
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What is the operon structure? |
Bacterial DNA, regulatory gene, promotor, operator, series of structural genes (code for enzymes of the same metabolic pathway), and represser protein. (Regulatory gene codes for represser protein, which goes to the operator.) |
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How does the Lac oepron work> |
The inducer (allolactose) binds to the active represser to make it inactive, and then the RNA polymerase goes and attaches to promotor and codes genes. This is induction. When lactose is gone, and the represser is active and binds on again, this is repression. |
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What is the TRP operon> |
E.coli operon, and makes the amino acid trypyophan. Default is ON, unless TRP is present. If TRP present, then repression takes place. TRP acts as corepressor. TRP binds to repressor and then they both attach to operon. When TRP isn't around, TRP then detaches and the repressor comes off too. This makes a derepressed state. |
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How does cAMP work with lactose? |
If glucose level is low, then cAMP is high and cAMP binds to CRP (cAMP receptor protein), and the complex activates the lac operon. |
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Explain the four situations of positive and negative control/ |
Lac high, glu high: allolactose is the inducer, cAMP is low, so CAP is inactive, Lac high, glu low: lac binds to repressor, and cAMP is high, so CAP binds to CAP site Lac low, glu high: negative control implimented, CAP inactive Lac low, glu low: neg and pos control in place. CAP is in place, but repressor is also in place. |
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What is transcription level control? |
Recombinent DNA methods to alter sequences, reductionist approach (what happens if we stop this gene?). You can tets this with hybridization using radioactive probe. But if there is an issue, use reporter gene. |
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How do you alter cloned DNA? |
You can use the deletion method by mapping where the TATA box is. You can also make just a small changw which is dite-directed mutagenesis. |
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How do you analyze the expression of the trasngene? |
Use a reporter gene. This means it is from another animal which usually isn;t in a mouse so you can't miss it. You can also add in a green fluorescent protein. |
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What happens if you delete some of the promotor genes? |
The TATA box makes it go down 60%, CAAT box goes down 50%, and GC box goes down 85%. Many promoters contain binding sites for a number of proteins that can influnce transcription.
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What are transcription factor types? |
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What are DNA binding and activation domains? |
DNA binding: sequence specific interaction which is palindromic and may form dimers. Activation: must interact with one or more proteins to promote transcription or repress it. |
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What is hetero vs homodimer? |
Homo: two identical molecules which get together. Hetero: Two different molecules which get together |
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What is combinational control? |
Two different proteins which are encoded by different genes, and recognize similar sequences. These two together can see 3 different sites. Site A, site B, and site AB. |
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What is microarray technology? |
Also called gene chip, requires knowledge of all genes. A robot synthesizes DNA samples for all genes. Lithography process stamps the DNA samples in a sequence so you know where everything is. Used to compare global gene expression patterns. |
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Why do nitrogenous bases have specific functional groups that project into the major groove? |
Sequence specificity for protein binding resides in the type and order of bases. Because it is large enough to fit transcription factor to bind to. |
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What is the zinc finger transcription factor? |
Zn coordinates binding 2 cysteines and 2 histidines. IT then makes a finger which fits into the major groove. |
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What si the helix-loop-helix TF? |
They bind as dimers. The basic domain interacts with DNA to determine sequence specificity. Often are heterodimers encoded by different genes. |
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What is the leucine zipper motif? |
LEU is found every 7th AA for about 40AA in an alpha helical sequence. The LEU are then on the same side of the molecule then, two get together and wind around each other to make a zipper. It shields them from water. |
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What is the bZIP TF? |
There is a coiled coil domain which is a LEU zipper, and there is also a basic domain which mediates DNA binding. |
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Activation vs Repression. |
If a cis-acting site has a bound molecule (activator), this may prevent the binding of another molecule (repressor). Often there is competition for binding between an activator protein and a repressor protein. Expression depends on the strength of binding and the number of + and - factors involved. |
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What is transcription profiling? |
A method of examining the expression of many or all genes simultaneously. You detect and quantify based on amount of fluorescently labelled cDNA that hybridizes to grid spots. |
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What are chromatin modifications? |
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What is heterochromatin? |
Remains condensed after mitosis, there are two types. Constitutive and facultative. |
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What is constitutive> |
Always condensed, such as repetitive DNAs of centromeres.
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What is facultative? |
Condensed depending on cell type, stage of development or other stimulus.
ex. X-chromosome inactivation. During embryogenesis, one X is inactivated. |
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What is euchromatin? |
Returns to dispersed state after mitosis takes place. |
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WHat is methylation? |
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What is acetylation? |
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What is alternative splicing? |
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What is a poly A tail? |
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What is proteasome? |
An intracellular recycling center, removes ubitquitin from protein, unwinds protein, cuts it up tp e
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What are epigenetics? |
Mechanisms that alter expression but which do nto involve changes to DNA sequence (no mutations), modifications of DNA and histones. |
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Explain calico cats. |
During embryogenesis, one X is inactivated. In calico cats, one X has black, other X has orange. There is an X-linked XIST gene responsible
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What are N-terminals modifided? |
The N-terminal part of the histone sticks out of the circle, and can get modified. |
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How is heterochromatinization driven by siRNAs? |
Transcription happens, and then this can form dsRNA. Then dicer comes in and cuts it up and keeps the guide strand of RNA to make siRNA. |
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What does SUV39H1 do? |
A histone methyl transferase modifies local histones to create a bidning site for HP1 (heterochromatin protein 1). |
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Where is heterochromatin in loop domains? |
Is the shrunk up version of the loop, insulators prevents spread of heterochromatin. |
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What is steroid hormone activation? |
Cortisol finds a clucocorticoid recpetor, and then together they go to the nucleus. This then finds promotors and turns on genes. Proteins are then made. |
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What is steroid induced gene activation? |
GR binds to GRE (clucocorticoid response element, is cis-acting). The CRB/HAT (histone acetyltransferase), opens up the histones and adds an aceytl group. This reduces + charge of LYS. Now that the histones are open, SWI/SNF opens the promotor, and RNA can come along. |
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How does SWI remodel complexes? |
Can work in 4 differnet ways. Can slide the gene so the TATA is exposed. Conformational change so the TATA box is less tightly wound. A histone exchange, or a histone dissociation. |
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What is a transcirptional repression? (revesring action of HATs) |
Repressor binds to active chromatin, co-repressor and HDAC recruited and deacetylated histones, HDACs: histone deacetylation complexes, now the chromatin is inactive, methylation by HMT maintains repressed state. |
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WHat is site selection for alternative splicing? |
SR is bind enhancers, and hnRNP is bind silencers. These proteins may recruit different exons by controlling the ability of spliceosome components to bind juncion sequences. The result is some weak splice sites bypassed, others are ignored. |
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What is alternative splicing? |
Producing multiple mRNAs from the same primary trnascript from exons pointing to areas to cut. Expands capacity of genome to encode a much larger proteome. |
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What happens in fertilization? |
Translation is enhanced by CPEB phosphorylation. Maskin which is a global inhibitor is displaced from CAP and releases eIF4E. Poly A poymerase elongates poly A tail as well. |
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What does ferritin do? |
Binds to iron and protects cell from toxic effects. Contains IRE (iron response element). The IRP (iron response element) covers the active site IRP and this prevents translation. When iron is around it binds to the IRE and then ferritin is made. |
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What is like a fuse to a bomb? |
The poly A tail. Poly A nuclease constantly cuts at the tail, and then a decapping enzyme comes along and takes the cap off so it can't be transcribed anymore. |
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What is the maskin link? |
The longer the tail is, the more mRNA, the more stable, the more protein made. |
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What does ubiquitin ligase do? |
Attaches to degraded protein, and takes it to proteasome to be destructed. |
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What is conservative vs semi-conservative? |
Conserve: The parental strands are together, adn the daughter strands are together in separate helices. Semi: There is an equal mixture of the parental strand and daughter strands in each helix. |
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What is dispersive? |
A replication mechanism where the parental and daughter strands are randomly intermixed with each other. |
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What is density label? |
Meselson and Stahl used this in their experiment with E.coli. They used N15 as their desntiy label. Over time, replication takes place, and then DNA is uniformly labeled with N. Then they added in N14 to test the different time points and weights. They found there were 3 types of DNA. Dispersive, conservative, and semi because they all had different lines. Light on top, heavy on bottom. |
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WHat is a topoisomerase? |
Cuts DNA fragments at one strand to eliminate torsional stress during replication. |
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WHat is gyrase? |
Same as topoisomerase.
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WHat is DNA polymerase? |
Can only add mucelotides to a 3' OH group and must have template to copy. Requires dNTP + polymer. Two phosphates are cleaved off, alpha phosphate is kept, and used to attach new NTP. Makes new DNA 5 to 3, but while traveling down the DNA 3 to 5. |
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What is the leading vs lagging stand? |
Continuous synthesis= leading Discontinuous= discontinuous |
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WHat are Okazaki fragments? |
Nicks between adjacent DNA segments. DNA ligase links fragments together |
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WHat is primase? |
Forms the 3' hydroxyl group on the lagging strand synthesized by RNA primer. |
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What did JH taylor do? |
He used BrdU so when the cells replicated, they had BrdU where thymidine would be. Thymidine is dark, and BrdU is light. So after 2 rounds of replication, one chromosome awas darj and one was light. |
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What are the different types of DNA polyermase? |
Pol III: elongates strand from primed sites. Pol I: Acts as repair enzyme to remove RNA primer and replaces NTP with dNTP. |
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What is helicase? |
Unwinds parental strands. Goes from 5 to 3. |
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What is primase? |
Initiates strands |
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WHat is primisome? |
Primase and helicase traveling close to each other down DNA |
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What s replisome? |
When the lagging strand is turned around 180 degrees so that Pol III can work in teh same direction on both strands |
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WHat is a sliding camp? |
DNA poly finishes O fragment, the clamp then lets go, and the Pol III lets go. Helicase is then activated and creates a short strand. The clamp and DNA Pol then reattach and proceed until they reach the previous RNA ending sequence. Then again and again. |
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What is fiber autoradiography? |
Cairns took cells and radioactive thymidine so they would go into DNA. PLace those cells on glass silde, then tipped it upwards, then squirt with detergnet. This broke the membranes, so whats in the nucleus flows down the slide. He then could see the radioactive DNA on the slide in straight lines. These are single chromatin fibers where replication had begun and gone bidirectional. |
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What is replication licensing? |
Origins in yeast are called ARS (autonomously replicating sequence). ORC (origin reconition complex) proteins bind to the ARS in cell. MCM proteins bind to form a prereplication complex. S phase initiated by protein kinase. MCM proteins become part of replication complex. MCM proetins released. MCM cannot relicense replication until nuclear membrane is disassembled. |
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WHat is SSDB? |
Single-stranded DNA binding protein, keeps single strands unwound. |
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What is a telomere? |
A series of short repetitive DNA sequences located at the ends of eukaryotic chromosomes. At 5' strand end of each newly synthesized strands. If there was too much telomere, then the ends of the strands would get shorter and shorter. At the end of DNA, a telomere bidns to an internal telomere repeat so it becomes loop. Length means cell age. |
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What is telomerase? |
An enzyme which replicates the end of DNA. Has an RNA compenent which is complimentary to the 3' end of the DNA. This makes the 3' end longer. Such as to give the enzyme the room to lay down RNA primase to extend the 5' end. |
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WHat is endo and exonuclease? |
Exo is when the DNA pol destroys the DNA by removing the primer. This goes in 5 to 3. Proofreading function goes from 3 to 5. Happens from a free end. Endo means it goes from the inside of the strand. |
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What is proofreading? |
When DNA pol goes backwards on teh DNA to check for errors, and then cuts teh errors out. This is activated by altered angles when there are mismatched regions. |
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WHat is spontaneous mutation rate? |
This is how often DNA pol makes mistakes and the cell doesnt repair it. |
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What is error vs mutation? |
Error is when there is a mistake of DNA pol while replicating. Mutation is when these errors are replicated many times and is permanent. |
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WHat is mismatch repair? |
Takes place in other lesions when there is a mismatch error. |
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WHat is a pyrimidine dimer? |
When ultraviolet light induces a second pyrimidine to attach adn this can cause errors and cancer. |
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What is NERP? |
Nucleotide excision repair pathway. Transcription coupled pathway ensures actively transcribed genes are a high priority for repair. A global pathway is slower mechanism for other parts of the genome. |
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What is glycosylase? |
Cleaves sugar/base bond to release mispaired base. |
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What is BERP? |
Base excision repair pathway which both pathways use enzymes to remove the area containing damage. Glycosylase cuts the base out, endonuclease acts at the site, then a new base it attached and DNA ligase acts. |
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What happens when telomerase is knocked out? |
Telomeres fuse together. This forms a dicentric chromosome. Microtubules then capture the teomeres, but if they are caught by different directions, then the chromosome breaks. |
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How do prokaryotes fix their strands? |
DNA methylation at GATC sites as something it needs to watch. |
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What is G1, S, G2, and M phases? |
G1: cell grows and carries out normal metabolism, organelles duplicate. S: DNA replication and chromosome duplication G2: Cell grows and prepares for mitosis. M: Mitosis (prophase, prometaphase, metaphase, anaphase, telophase, cytokensis). |
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What is cyclin? |
Slowly increases as the cell cycle goes on. STarts at G1, then peaks at M. |
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What is cyclin-dependent kinase? |
Phosphorylate multiple target molecules altering their activities and controlling cell cycle progression. |
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What is MPF? |
Maturation promoting factor made of cyclin and cyclin-dependent kinase. Spikes during mitosis. |
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How do you determine the duration of cell cycle stages? |
Label with radioactive dNTP for short time. You will see the raioactivety in S phase. |
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What happens in prophase? |
Chromosomes condense, cytoskeleton disassembles, golgi and ER fragment, nuclear envelope breaks down. |
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What happens in prometasphase? |
Microtubules attach to chromosomes, chromosomes are moved to equator. |
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What happens in metaphase? |
Chromosomes are aligned and attaches to microtubules to both poles. |
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What happens in anaphase? |
Centromeres split, chromosomes go to opposite poles. |
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What happens in telophase? |
Chromosomes cluster, become dispersed, nucelar envelope assembles, golgi and ER reform. |
