Term
| The end product of gene expression is usually a _____ |
|
Definition
|
|
Term
| The end product of gene expression must be _____ |
|
Definition
|
|
Term
| First step of gene expression: _____ |
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Definition
|
|
Term
| Are all genes expressed all the time? |
|
Definition
|
|
Term
| ______ chews up mRNA (usually from the 5' end) |
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Definition
|
|
Term
| When & why is a cap put on the end of RNA? |
|
Definition
When: after some part of the RNA is made but the rest is being made
Why: To confuse the exonuclease |
|
|
Term
| Does non-messenger RNA get caps? |
|
Definition
|
|
Term
| Do most eukaryotic RNA have introns? |
|
Definition
| yes; intron-containing mRNA is called "pre=mRNA") |
|
|
Term
| 3 steps of gene expression that occur in the nucleus |
|
Definition
1. transcription
2. capping
3. slicing |
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|
Term
| ______ free up the DNA from the chromatin to express a gene |
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Definition
|
|
Term
| 2 things added when DNA --> RNA |
|
Definition
1. a cap
2. a polyadenylate tail |
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|
Term
| Where does cap-RNA-A splice out introns? |
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Definition
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|
Term
| Cap-RNA-A gets exported from nucleus into the cytoplasm...is this reversible? |
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Definition
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Term
|
Definition
1. translated by ribosomes
2. stored
3. T.O. (degraded) |
|
|
Term
| Can gene expression be regulated post-translation by protein co-factors? |
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Definition
|
|
Term
| 2 ways co-factors can affect a protein's activity |
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Definition
1. some proteins need co-factors to become active
2. some co-factors repress a protein's activity |
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|
Term
| intracellular localization |
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Definition
| how proteins get where they need to go in order to do their job |
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|
Term
| How does intracellular localization happen for proteins? |
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Definition
| there are molecules that can traffic them around |
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Term
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Definition
| something added to proteins that affects their solubility, localization, etc. |
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Term
| What often happens to secreted proteins as they go through the ER? |
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Definition
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|
Term
| How does phosphorylation affect the activity of a protein? |
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Definition
| Adding a P can affect the activity of proteins positively or negatively |
|
|
Term
| Can methylation be undone? |
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Definition
| Yes; there are also de-methylases |
|
|
Term
| ______ get acetylated to affect transcription |
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Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
| if proteins are misfolded, this can happen to the protein so it is degraded by a proteasome |
|
|
Term
| Are there many different kinds of PTMs? |
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Definition
|
|
Term
| Can mRNA be translated in storage particle? |
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Definition
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|
Term
| example of mRNA being stored for later use |
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Definition
| mRNA in eggs does not translate until it is fertilized |
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|
Term
| are translation & storage reversible? |
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Definition
| yes; they are reversible between each other |
|
|
Term
| example of a storage particle |
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Definition
|
|
Term
| second use of storage particles (beyond storage) |
|
Definition
| can also transport the mRNA to where it needs to be |
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|
Term
| mRNA can be stored as an _____ |
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Definition
|
|
Term
| during transport of mRNAs from one cell into another cell, it must be protected from ______ |
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Definition
|
|
Term
| what would happen if an intron is supposed to be removed because it has a STOP codon & it isn't? |
|
Definition
| that RNA can be subject to degradation |
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|
Term
| Where does turnover happen in the gene expression pathway? |
|
Definition
| can happen anywhere if something goes wrong |
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|
Term
| ______ surveil the cell for mistakes |
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Definition
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|
Term
| If any one of the steps in the gene expression process goes wrong, _______ |
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Definition
| the mistake will be recognized & degraded by the cell |
|
|
Term
| when can cells block gene expression? |
|
Definition
|
|
Term
| is regulation of gene expression positive or negative? |
|
Definition
|
|
Term
| constitutive gene expression |
|
Definition
|
|
Term
| inducible gene expression |
|
Definition
|
|
Term
| repressible gene expression |
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Definition
|
|
Term
| ubiquitous gene expression |
|
Definition
| expressed at all places at all times |
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|
Term
| temporal/spatial specific gene expression |
|
Definition
| expressed at a specific time or place |
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|
Term
| gene expression regulated by signals |
|
Definition
|
|
Term
| 3 ways we study the pattern of temporal & spatial specific gene expression that dictates the life of every organism we know |
|
Definition
1. RNA seq (transcription measurement)
2. ribosome profiling (translation)
3. mass spectrometry (protein) |
|
|
Term
| 3 things to study when discovering how the patterns in gene expression happen |
|
Definition
1. genes
2. cis elements
3. trans-acting factors |
|
|
Term
| 6 things we knew about before cloning (60s) |
|
Definition
1. chromosomes
2. DNA replication
3. chromatin
4. histones
5. transcription
6. translation |
|
|
Term
| 7 things they did not know about before cloning |
|
Definition
1. cloning
2. sequencing DNA
3. miRNAs
4. IncRNAs
5. transport
6. localization
7. epigenetics |
|
|
Term
| why can't you measure the molecular weight of the genome by gels? |
|
Definition
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|
Term
| 4 steps in the physicist solution to how big is the genome |
|
Definition
1. cut the genome into pieces
2. heat it to denature it
3. lower the temperature (or salt concentration) so that DNA will renature
4. look at the rate (how long it takes) for them to base pair back again (renature) |
|
|
Term
put these model organisms in hierarchy:
1. e. coli
2. drosophila
3. humans
4. mice
5. sea urchins
6. worms (c. elegans)
7. yeast |
|
Definition
1. e.coli
2. yeast
3. worms (c. elegans)
4. drosophila
5. sea urchins
6. mice
7. humans |
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|
Term
| 2 problems with running a 2D gel to determine how many genes you have (a gene makes proteins) |
|
Definition
1. some proteins will not be detectable because they are in such low concentration
2. some proteins will overlap in one spot on the gel because there is not enough space |
|
|
Term
| make a DNA copy of RNA using ______ (making radioactive cDNA) |
|
Definition
|
|
Term
| How many genes are expressed in one typical cell? |
|
Definition
| For higher eukaryotes, the number is around 10,000 |
|
|
Term
| 2 types of difference in the expression of genes in different kinds of cells |
|
Definition
|
|
Term
| In order to understand a disease state, you have to know ______ |
|
Definition
| why/how something works correctly |
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|
Term
| 3 things scientists use to decide upon which of the thousands of genes to study |
|
Definition
1. keep it simple/easy to study
2. needed it to be interesting (ex. spatial or temporal specific, something that responds to a signal, medically relevant/relevant to the human condition)
3. needs to be representative/a useful example |
|
|
Term
| first 3 models to study gene expression before cloning |
|
Definition
1. globin
2. ovalbumin
3. adenovirus |
|
|
Term
| How did they study gene expression without cloning? |
|
Definition
| you need a reporter assay for mRNA (ribosomes could be purified & RNA could be extracted from sample --> RNA would be added to ribosomes & you could see if your desired protein was created or not) |
|
|
Term
| first step to studying gene expression |
|
Definition
| find a model system that is easy, representative, interesting, etc. |
|
|
Term
| standard view on model organisms |
|
Definition
| each model organism is good for certain things & no model organism is good for everything |
|
|
Term
| What leads to a good model system? |
|
Definition
| a lot of knowledge about a gene product --> whether expression might be "interesting" |
|
|
Term
|
Definition
| purifying a gene from any organism |
|
|
Term
| 4 model organisms mostly used before cloning |
|
Definition
1. yeast
2. mice
3. rats
4. drosophila |
|
|
Term
|
Definition
1. cut & purify gene sized pieces out of the genome
2. insert the pieces into cloning vectors (plasmids, lambda phage, YACs, cosmids, etc.)
3. identify your desired gene among the mixture |
|
|
Term
| how to get gene sized pieces |
|
Definition
| genome gets sheared into the pieces; restriction enzymes cleave DNA & leave sticky ends |
|
|
Term
| 3 difficult steps in cloning |
|
Definition
1. generate gene-sized pieces from the genome
2. separate the gene-sized pieces from each other
3. how do you know which colony contains the desired gene |
|
|
Term
| problem with using 4 base cutters in cloning |
|
Definition
| they make blunt ends not sticky ends |
|
|
Term
| Maniatis used ______ for his cloning vector |
|
Definition
|
|
Term
| How do you get your libraries from bacteria? |
|
Definition
| can spin down bacteria that contain your plasmids in a tube |
|
|
Term
| A tube of lambda particles in liquid would have ______ DNA |
|
Definition
|
|
Term
| What does it mean that identifying which colony or plaque contains your desired gene is "idiosyncratic"? |
|
Definition
| how to find your desired gene depends on what you know about that gene/its expression/its product |
|
|
Term
| genetic approach for identifying which colony or plaque contains your desired gene |
|
Definition
| could screen for mutants (having mapped the mutant allele via genetic crosses) |
|
|
Term
| biochemistry approach for identifying which colony or plaque contains your desired gene (if you have purified the desired protein) |
|
Definition
| can parse out its amino acid sequence or have an antibody for it or have an activity assay (if it is an enzyme) |
|
|
Term
| When would you identify desired genes using expression pattern? |
|
Definition
| best for when you are not interested in one gene but instead are interested in any gene that has a specific expression pattern (ex. only expressed in the brain & not elsewhere in the organism) |
|
|
Term
| Why would different fragments of DNA require different conditions to split apart? |
|
Definition
| C-G is a triple hydrogen bond vs. A-T is a double |
|
|
Term
| nucleic acid hybridization assay |
|
Definition
| after heating the DNA so it comes apart, pipette it onto a piece of paper --> in a tube, add radioactive DNA & see if it will base pair with the regular DNA fragments on the paper in the tube (ex. radioactive Watson fragment to "cold" Crick fragment) |
|
|
Term
| Can there be noncomplete base pairing (nucleic acid hybridization assay)? |
|
Definition
|
|
Term
| Ed Southern cut plasmids with the gene with ______ |
|
Definition
|
|
Term
|
Definition
| take the gel after you run it, drop it in a tray of denaturant (fragments will separate within each DNA section), transfer the DNA out of the gel to the paper ("dot blot" protocol idea) |
|
|
Term
| The northern blot is doing what Ed Southern did but with ______ |
|
Definition
|
|
Term
| what does a northern blot tell you? |
|
Definition
| whether (using a probe) there is an RNA that is complementary to/will base pair with/is made from the DNA probe |
|
|
Term
| What would it mean if an RNA band on northern blot only shows up in brain cells & not liver cells? |
|
Definition
| this gene must be brain specific |
|
|
Term
| What would it mean if thicker RNA band on northern blot in brain cells than liver cells? |
|
Definition
| even though the gene is present in both cell types, it is more prevalent in brain cells |
|
|
Term
| 3 things you can tell from a northern blot |
|
Definition
1. whether RNA is expressed
2. how much if it is expressed (relative to other types of cells)
3. approximate size (because you run with molecular weight) |
|
|
Term
| 2 things it could mean if two RNA on a northern blot show up in the brain but only one matches with the liver |
|
Definition
1. differential splicing (tissue-specific)
2. both are made but one is degraded in the liver (turnover) |
|
|
Term
|
Definition
1. taking double stranded DNA
2. melting it
3. putting it on a piece of paper where it can hybridize with an mRNA |
|
|
Term
| 4 steps to southern blots |
|
Definition
1. cut DNA with restriction enzymes to make a bunch of restriction fragments
2. separated the fragments on a non-denaturing gel to separate on basis of length
3. THEN denature & transfer them to paper (mimicking a dot blot) & get to hybridize with "hot" probe
4. exposing it to x-ray film makes radioactive line show up as black line on the gel |
|
|
Term
| range of DNA size that can work with southern blot |
|
Definition
| plasmid or phage DNA OR with the entire genome |
|
|
Term
| Why do northern blots separate RNA on a denaturing gel? |
|
Definition
| denaturing gel prevents RNA from base pairing into helices with itself |
|
|
Term
| the northern blot is the same idea as the southern blot but just with _____ |
|
Definition
|
|
Term
| can you assay splicing with a northern blot? |
|
Definition
|
|
Term
|
Definition
1. separate proteins on a denaturing gel
2. plot to paper
3. probe with antibodies |
|
|
Term
| can probes be tissue-specific? |
|
Definition
|
|
Term
| how was reverse transcriptase discovered? |
|
Definition
| the enzyme that viruses use to cause tumors by turning their RNA to DNA |
|
|
Term
| what will you see if a gene is expressed in both brain & liver tissue but much more in one vs the other |
|
Definition
| you will see a darker signal with the probe that was abundant vs the one that was not abundant |
|
|
Term
| 3 proof of principle experiments done by Maniatis (having built these libraries, can we demonstrate utility of them?) |
|
Definition
1. globin genes: used a very enriched cDNA (red blood cells, size screen)
2. chorion (silkworm eggshell) genes: extract RNA from that tissue, make cDNA, probe libraries --> end up with chorion specific genes
3. drosophila developmental stage: did not care what the gene was but wanted them temporally specific to start of drosophila experiment |
|
|
Term
| cloning of genes lead directly to the discovery of ______ |
|
Definition
|
|
Term
| How does sequencing help with necessary & sufficient studies? |
|
Definition
| ability to delete out specific sequences & see how that affects transcription/translation |
|
|
Term
| what had to happen to dot blots, northern blots, & southern blots to look at the entire 'ome? |
|
Definition
|
|
Term
|
Definition
| entire set of sequences in the studied chromosomes |
|
|
Term
|
Definition
| study of the entire genome |
|
|
Term
|
Definition
| the development & systematic application of experimental methodologies to analyze gene functions on a genome-wide scale |
|
|
Term
|
Definition
| the sequence & amounts of all RNAs in a sample |
|
|
Term
|
Definition
| the study of the transcriptome |
|
|
Term
|
Definition
|
|
Term
|
Definition
| same as functional genomics except it is the function of proteins not genes |
|
|
Term
|
Definition
| all of the interactions among all of the molecules in your sample |
|
|
Term
|
Definition
| all of the metabolites in a given sample |
|
|
Term
| were all genes discovered by cloning? |
|
Definition
|
|
Term
|
Definition
| sequences in the genome that don't encode genes |
|
|
Term
| are groups of genes that functionally work together linked together physically? |
|
Definition
|
|
Term
| what controls a gene's expression within the genome? |
|
Definition
|
|
Term
| 3 parts of the ORF (sequence of codons that code for an amino acid chain) |
|
Definition
1. starts with a start codon (usually ATG in DNA; AUG in RNA)
2. follows with continuous codons
3. ends with stop codon |
|
|
Term
| 3 things that can be "wrong" about a start/stop codon when identifying genes |
|
Definition
1. non-canonical start codons
2. non-canonical stop codons
3. stop codons that ribosomes skip |
|
|
Term
| are introns usually involved in genes? |
|
Definition
|
|
Term
| another way you can find a gene besides looking for an ORF |
|
Definition
| look for a coding sequence that codes for a conserved protein across species (homology) |
|
|
Term
| another way you can find a gene besides ORF or homology |
|
Definition
| look for RNA (definition of a gene is something that is transcribed into an RNA) |
|
|
Term
| where did genomics start? |
|
Definition
|
|
Term
| ______ helped to fill in the blanks between mapped genes when many different genes had been mapped to the yeast chromosome |
|
Definition
|
|
Term
| 4 things you need to make an artificial chromosome |
|
Definition
1. centromere
2. telomeres
3. gene
4. origin(s) of replication |
|
|
Term
| what is the shape of an artificial chromosome? |
|
Definition
|
|
Term
| how was the problem of artificial chromosomes being initially too short to behave like a chromosome solved? |
|
Definition
| jammed DNA into cloning site --> caused it to act more like chromosome |
|
|
Term
| which genome served as a proof of principle for other organisms genome sequencing? |
|
Definition
|
|
Term
| Do yeast genes typically have introns? |
|
Definition
|
|
Term
| one surprising discovery from yeast genome sequencing |
|
Definition
| many of the genes that had been discovered in humans (especially where mutations cause disease) had homologues on yeast! |
|
|
Term
| what do you learn from running dot blot/northern blot with regard to time? |
|
Definition
| can see when the mRNA is available at certain times to base pair with the radioactive DNA |
|
|
Term
| If you wanted to do similar to dot blot/northern blot but genome-wide or transcriptome-wide, you would need to scale up to do ______ |
|
Definition
|
|
Term
| 5 steps for transcriptomics |
|
Definition
1. PCR out the various DNAs
2. heat them up so Watson & Crick come apart
3. pipette onto tiny glass slides
4. hybridize it to various labelled DNAs
5. can see how RNA expression is different under various conditions |
|
|
Term
| 6 steps for dot blot & filter hybridization |
|
Definition
1. PCR a specific gene when you know where it is in the sequence/its sequence
2. take the PCR fragment & heat it to separate Watson from Crick
3. pipette it onto a piece of paper
4. isolate mRNA from cell (i.e. brain or liver etc.) & make cDNA copies of all the RNAs
5. put cDNAs in beaker with the paper
6. look for which genes are labeled by which fluorescent |
|
|
Term
| dot blots morphed into microarrays by ______ |
|
Definition
| pipette PCR fragments from every gene in the genome onto one glass slide |
|
|
Term
| solving the resolution problem of dot blots/microarrays |
|
Definition
| gene chips method --> make DNA fragments from entire genome (as opposed to PCR fragments) |
|
|
Term
| solving the gene chips method lots to make problem: _____ |
|
Definition
|
|
Term
| 3 things you get with RNA-Seq |
|
Definition
1. the RNAs
2. where they start
3. where they stop (assuming the transcriptase makes it all the way to the end) |
|
|
Term
| If you don't see something in a probe does that mean it isn't there? |
|
Definition
| no! you can say it is not detecable |
|
|
Term
| What kind of RNA should not have introns? |
|
Definition
|
|
Term
| ______ provided the necessary foundation for discovering the transcriptome |
|
Definition
|
|
Term
| 2 steps to studying the transcriptome |
|
Definition
1. population of RNAs
2. make cDNAs from them by using reverse transcriptase & primer |
|
|
Term
| problem you could have using reverse transcriptase |
|
Definition
| if you use an oligiodT primer, there might be a secondary structure in the RNA which may halt the reverse transcriptase before it gets to the end |
|
|
Term
| benefit of using random hexamer for transcriptomics |
|
Definition
|
|
Term
| 3 steps to RACE (rapid amplification of cDNA ends) |
|
Definition
1. have a primer of a sequence that is complementary of your RNA
2. ligate an oligo at the end of your RNA
3. make a DNA that primes to that end & it will move along until it gets to the primer bound to the RNA |
|
|
Term
|
Definition
| you get a readout of what is present in the cell at that time |
|
|
Term
| gaps between ORFs --> discovery of ______ |
|
Definition
|
|
Term
| What taught us about introns? |
|
Definition
|
|
Term
| Can the sequence of RNA start before ORF & end after ORF? |
|
Definition
|
|
Term
| short ORFs (uORFS or "upstream ORFs") |
|
Definition
| used to regulate the translation of the downstream ORF in the very same transcript |
|
|
Term
| can you have overlapping transcripts? |
|
Definition
|
|
Term
|
Definition
| a transcript from both Watson & Crick in the same cell (one reads --> & the other reads <--) |
|
|
Term
| can a transcript overlap an intron? |
|
Definition
|
|
Term
| why is it hard to perfectly predict the proteome from the transcriptome? |
|
Definition
| hard to say exactly what conditions cause the varying ways a transcript can be read |
|
|
Term
| is the proteome a steady state readout? |
|
Definition
|
|
Term
| 3 ways to find the proteome |
|
Definition
1. genome out
2. transcriptome out
3. proteome in |
|
|
Term
| both transcriptome & proteome are steady state....so how do you find out what is actually being made in the cell being studied? |
|
Definition
|
|
Term
|
Definition
| an mRNA being translated will be loaded up with ribosomes --> measure the density of ribosomes on the mRNA |
|
|
Term
| how precise is ribosome profiling? |
|
Definition
| tells you down to which reading frame is being used |
|
|
Term
| 4 steps to ribosome profiling |
|
Definition
1. isolate mRNA attached to ribosomes
2. treat with nuclease
3. RNA covered up by ribosomes will be protected (ribosomes that are naked will be eaten by the nuclease)
4. sequence the RNA that was protected |
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|
