Exam 1 Flashcards
describe Western Blotting (what is it, what is it used for, positive or negative controls, etc.)
- allows for the presence or absence of a particular protein to be detected within a specific tissue
- uses 1D gel electrophoresis
- steps: sample preparation, SDS page, transfer of protein, blocking, incubation with primary and secondary antibodies and detection of target protein
- positive control: tissue that is known to express the protein you are looking for
describe Southern Blotting (what is it, what is it used for, positive or negative controls, etc.)
- allows for the study/analysis of a single genes, specifically the structure of different genes in different cell types
- electrophoresis of DNA that has been cut by a restriction enzyme, run through an agarose gel, then transferred to a nitrocellulose filter, where hybridization occurs and the probe detects homologous DNA
describe Northern Blotting (what is it, what is it used for, positive or negative controls, etc.)
- allows for the study of a single gene (RNA)
- the RNA extracted from a particular tissue is electrophoresed on an agarose gel, transferred to a nitrocellulose filter, and hybridized to a radioactive probe derived from the gene encoding the mRNA of interest
- something that you know contains the RNA sequence that you are looking for in your Northern blots
what is the probe in Western blotting?
antibody
what happens in first dimension isoelectric focusing?
protein’s charge influences migration
what happens in second dimension isoelectric focusing?
protein’s mass influences migration
why is it beneficial to run a 2D gel after a 1D gel?
the proteins will move to a position in the gel based on both their size and charge, allowing for much greater resolution and allows a number of differences in protein composition of particular tissues to be identified
define proteomics
large scale study of proteins
define transcriptomics
study of the transcriptome (set of all
RNA molecules in cells or tissues)
what is the probe in Northern blotting?
radiolabeled DNA
what is the probe in Southern blotting?
radiolabeled DNA
what is on the membrane in a Western Blot? Northern? Southern?
- W: protein
- N: RNA
- S: DNA
describe the technique of RT/PCR (what is it, how is it done, why is it useful)
- reverse transcriptase polymerase chain reaction
- RT reverse transcribes mRNA to DNA called cDNA, this cDNA is then specifically amplified by hybridization with complimentary primers and DNA synthesis by DNA polymerase enzyme
- is more sensitive than Northern blots
- very rare transcript abundance can be measured
describe the gene chip technique (what is it, how is it done, why is it useful)
- gene chip analysis allows for/combines the ability to look at variation in the total RNA population of different tissues with the ability to look specifically at the variation of specific mRNA **mRNA expression patterns in different tissues
- a gene chip is prepared containing sequence information from a wide variety of RNAs (done either by spots of cDNAs or oligonucleotides), then hybridize with fluorescent sequences prepared from all the mRNAs in an individual tissue (if mRNA is present in a tissue, the chip will fluoresce, with the signal being proportional to the amount of RNA present)
- usefulness: can spot out many different DNA sequences onto a very small chip and obtain a lot of data, shows qualitative and quantitative differences in the mRNA populations of different tissues
what are the negatives of gene chip analysis?
- microarrays can be biased because someone has to choose what to place on the array
- cross hybridization can occur
- difficulty quantifying highly or lowly expressed genes
describe RNAseq
- technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also known as transcriptome
- allows researchers to detect both known and novel features in a single assay
what did Southern blotting help to prove, or rather disprove?
- disproved the DNA loss model (where scientists thought that gene expression differences were due to DNA loss) as well as the DNA amplification model and rearrangement model
- it did this by showing that specific DNA bands are still present even in a tissue where the gene is not expressed (DNA loss model), and the bands do not become more intense in tissues with expression (DNA amplification model) and there is no difference in the size of the band between different expressing tissues (DNA rearrangement model)
what are totipotent cells?
cells that can give rise to all the cell types in an adult organism
what conclusion did experiments with totipotent cells help scientists come to?
irreversible changes to DNA are not responsible for control of gene expression during differentiation
describe isoelectric focusing
the separation of proteins based on their charge
describe SDS-PAGE
an electrophoresis method that allows protein separation by mass
describe the pulse labeling technique (what is it, how is it done, why is it useful)
what is a limitation of it?
- used to assess the transcription rate of a specific gene (gene A) by measuring the amount of radioactivity (dots) incorporated into nascent transcripts
- first, nuclei is isolated, then radioactive nucleotide is added, allowing for transcription to occur, incorporating the radioactive NT to the nascent RNA chains, followed by hybridization to DNA of gene A
- provides the most direct means of measuring transcription
- limitation: brief labeling times, so primarily target highly expressed RNAs (rare RNAs don’t incorporate enough to label to become detectable)
describe the nuclear run-on assay technique (what is it, how is it done, why is it useful)
allows transcriptional control to be demonstrated for a wide variety of genes (more sensitive than pulse labeling because more radioactivity gets into cell)
describe micrococcal nuclease digestion
- mild DNA digestion enzyme
- targets linker DNA between nucleosomes
- organizes DNA into nucleosomes
describe DNaseI hypersensitive analysis
- used for detecting DNase1 hypersensitive sites
- isolate chromatin
- digest with very small amount of DNase1
- purify partially digested DNA by removing protein
- digest with restriction enzyme and carry out Southern blotting, probing for gene of interest
- monitor appearance of specific smaller band due to presence of DNase1 hypersensitive site within the DNA being tested
describe chromosome immunoprecipitation
- ChiP
- chromatin fragments from a sample are purified. antibodies then specifically recognize methylated C residues and are then immunoprecipitated, allowing for analysis by high-throughput DNA sequencing
sodium bisulfite treatment vs sodium butyrate treatment
- sodium bisulfite: takes unmethylated cytosine residues and converts them to uracil
- sodium butyrate: inhibits a cellular deacetylase activity, therefore increasing acetylation
describe mapping global DNA methylation patterns and histone modifications
- split chromatin sample
- do ChIP assay on one and ChIP + sodium bisulfite on another
- for the ChIP assay, look at DNA modification pattern. for the ChIP + SB, look at the DNA methylation pattern
- through computational methods, create map comparing histone modifications and DNA methylation patterns across entire genome
what is significant about a puff on polytene chromosomes?
they represent areas of intense transcriptional activity and can be directly visualized via radiolabeled nucleotides
in what ways do regulatory RNAs influence gene expression?
- can block transcription (negative regulation)
- can amplify transcription (positive regulation)
what type of regulatory RNAs exist? how are they synthesized?
- miRNAs: synthesized from a single-stranded RNA that has formed a hair loop
- siRNAs: processed from a dsRNA precursor
what is chromatin?
a complex of DNA and proteins, can influence gene expression
compare open and closed chromatin and the structure of open and closed chromatins
- open = active, euchromatin
- closed = inactive, heterochromatin
what influences chromatin structure?
histone post translational modifications (HPTMs), DNA methylation, histone variants, remodeling enzymes, and effector proteins
what enzymes are capable of modifying histones?
histone acetyl transferases (HAT), histone deacetylases (HDAC), histone methyl transferases (HMT), histone demethylases (HDM) and TET enzymes
how are DNA methylation patterns maintained?
- maintenance methylation: Dnmt1
^ recognizes hemi-methylated duplexes
^ does not methylate unmethylated sites - De novo: Dnmt3a or Dnmt3b
^ target only fully unmethylated C-G duplexes
how are DNA methylation patterns modified?
- enzymes such as HDAC, HMT, TET and HAT as well a ubiquitination and MeCP2 proteins
- active demethylation
- passive demethylation
what factors does HP1 interact with? what does HP1 do?
- binds to methylated lysine 9 residues
- recruitment of HP1 results in tightly packed organization/structure (heterochromatin)
- can also recruit an HMT enzyme, promoting the methylation of K9 on adjacent chromosomes, leading to more heterochromatin/tight packing of chromatin
what histone modification promotes HP1 interaction with histones? which histone does HP1 interact with?
- methylation of K9
- H3
describe Dnmt1
- maintenance methylation
- recognizes only hemi-methylated sites (only one C is methylated) and methylates the second site
what is ecdysone and does ecdysone treatment do?
hormone, triggers transcription
what is the function of miRNAs? does it require Dicer protein for maturation? single or double stranded RNA?
- function: control of cellular gene expression
- require Dicer: yes
- ssRNA
what is the function of siRNAs? does it require Dicer protein for maturation? single or double stranded RNA?
- function: control of viral and cellular gene expression
- require Dicer: yes
- dsRNA
what is the role of Dicer in regulatory RNAs?
- for miRNA, it binds and then cuts the ssRNA portion of hair-pin loop, releasing the dsRNA pieces with one being degraded and one forming the miRNA
- for siRNA, it binds to the dsRNA and then cleaves it into smaller dsRNA molecules, and one of these forms the siRNA
what did scientists initially characterize the production of siRNAs as?
a defense mechanism against invading viruses
define transgene
a gene from one source that has been incorporated into the genome of another organism
define promoter
a sequence of DNA needed to turn a gene on or off. the process of transcription is initiated at the promoter. usually found near the beginning of a gene, the promoter has a binding site for the enzyme used to make an mRNA molecule
transgene inserts can lead to formation of _______
siRNAs
TP = ______
CP = ______
- transgene promoter
- cellular promoter
pseudogene expression can lead to formation of ______
siRNAs
what is a pseudogene?
a false gene/a gene that does not code for a functional protein
how can/do transgene insertions lead to formation of siRNAs?
- transgene inserts itself into host genome close to a cellular promoter
- CP transcribes transgene in antisense orientation
- this antisense RNA transcript can then bind to the normal sense RNA transgene transcript
- dsRNA
- dsRNA will bind a Dicer protein
- Dicer cuts up the dsRNA and produces siRNA which can bind both the sense and antisense of the transgene
how can/do the expression of pseudogenes lead to formation of siRNAs?
- pseudogene is transcribed to produce an antisense transcript
- antisense transcript can hybridize to the protein-encoding region of the sense transcript of the functional gene
- this serves as substrate for siRNA
what are lncRNAs? what do they do?
-generally defined as transcripts more than 200 nucleotides that are not translated into protein
- can regulate gene expression in two ways:
- altering chromatin structure by recruiting RF that promotes heterochromatin
- competition for regulatory proteins (regulatory proteins might “prefer” lncRNAs and then activation of protein-coding gene isn’t occurring)
describe histones H2A, H2B, H3 and H4 (type and molar ratio)
- H2A: slightly lysine rich, molar ratio of 2
- H2B: slightly lysine rich, molar ratio of 2
- H3: arginine-rich, molar ratio of 2
- H4: arginine rich, molar ratio of 2
describe the histone fold
- 3 alpha helices
- allows heterodimer formation
why is the micrococcal nuclease technique important?
allows for the visualization of the fact that DNA organizes into nucleosomes
the term beads on a string describes
the organization of DNA into nucleosomes joined by visible linker DNA
beads on a string constitutes what stage of the packaging of DNA?
the first
describe chromatin remodeling complexes (examples, what they do, how they do it, etc.)
- examples: SWI/SNF , ISWI
- remodels chromatin via ATP hydrolysis (each complex contains an ATPase)
what are the chromatin remodeling outcomes?
- DNA becomes exposed
- nucleosome displacement
- complete nucleosome removal
how do you determine where nucleosomes bind DNA?
micrococcal nuclease digestion and immunoprecipitation
- cross-link nucleosomes to DNA with formaldehyde
- lyse cells and isolate chromatin
- digest with micrococcal nuclease and purify nucleosomes
- isolate nucleosome-associated DNA and purify 200 base pair mono-nucleosomal DNA by gel purification
- identify DNA which was associated with nucleosomes in the intact cell by DNA sequence analysis
chromosome remodeling can result in…
- a change in nucleosome structure
- the displacement of a nucleosome along the DNA
- the complete removal of a nucleosome
what is a more precise method of analyzing the position of nucleosomes? how is it done?
- chemical modification of histone H4
- attach a reactive group to H4, converting the serine at position 47 to a cysteine
- when exposed to a mixture of copper and hydrogen peroxide, hydroxyl radicals that cleave the DNA of adjacent bases to the nucleosome
- DNA fragments are generated, allowing for analysis of the nucleosome position
what are some examples of histone interactions that post-translational modifications that can influence?
- DNA
- other histones
- regulatory proteins
post translational modifications target what part of the histone?
the amino terminus
what is acetylation?
the free amino group on specific lysine residues is modified by one of the hydrogen atoms being substituted by an acetyl group (OCH3)
how does acetylation affect the net charge of lysine?
charge reduction
____________ catalyze the acetylation of histones
HATs (histone acetyltransferases)
what is a coactivator protein?
a type of transcriptional coregulator that binds to an activator (a transcription factor) to increase the rate of transcription of a gene
___________ removes the acetyl group from lysine amino acids
HDACs
what is methylation?
the replacement of 1, 2 or 3 hydrogen(s) with a methyl group/methyl groups to form methyl-lysine, dimethyl-lysine or trimethyl-lysine
can methylation target arginine residues? can acetylation?
methylation can, acetylation cannot
how does methylation affect the net charge of lysine?
no effect
what is special about lysine 9?
it can be methylated and acetylated
explain how DNA deletion events are different from DNA methylation events and why this is important
- DNA deletion events are irreversible, while methylation events are reversible
- methylation patterns being reversible shows the stable but not irreversible nature of the differentiated state
a defect in the ________ protein might lead to enhanced acetylation of H3
MeCP2
what is the MeCP2 protein/what does it do?
- a protein that binds directly to methylated CG but not unmethylated
- results in tightly packed chromatin structure and transcription repression
what are two results of K9 methylation?
- blocked acetylation at K9
- inhibited acetylation at K14
what is the result of K9 demethylation?
acetylation at K14
describe ubiquitination (where does it occur, what does it target, what happens to the net charge)
- occurs only on H2A and H2B
- targets one K only
- reduces positive charge
describe the result of ubiquitination of H2B
- enhances methylase accessibility and recruitment
- leads to methylation of H3
describe acetylation (effect on net charge, which amino acids and histones it occurs on)
- reduction in net positive charge of histone
- amino acid: K
- histone(s): core
describe methylation (effect on net charge, which amino acids and histones it occurs on)
- no reduction in positive charge
- amino acid(s): K and R
- histone(s): core
describe ubiquitination (effect on net charge, which amino acids and histones it occurs on)
- reduction in net positive charge of histone
- amino acid(s): K
- histone(s): H2A or H2B
describe phosphorylation (effect on net charge, which amino acids and histones it occurs on)
- reduction in net positive charge of histone
- amino acid(s): S and T
- histone(s): core
contrast a genetic and an epigenetic change
- genetic: a change in DNA sequence, irreversible
- epigenetic: change in modification of DNA or protein, reversible
what are two pieces of evidence that show that actively transcribed genes are organized into nucleosomes?
- beads-on-a-string structures are visible before and after RNA polymerase
- DNA of active genes is still organized into nucleosome ladders detectable by micrococcal nuclease digestion
enhanced DNase1 sensitivity indicates chromatin is not in the ________ nm fiber structure
30
what sets the stage for transcription to occur?
transition to an open chromatin structure
describe three factors of commitment to a differentiated state and remembrance
- maintenance of a differentiated state in response to a stimulus
- cells remember previous phenotype in absence of the stimulus
- return to phenotype in response to stimulus
_________ are a good model for studying cell fate
imaginal discs
what did the experiments with imaginal discs show?
irreversible mechanisms such as DNA loss can’t explain commitment, but chromatin can!
what is special about phosphorylation?
it targets serine or threonine residues, not lysine
what is special about histone variant H2AZ?
- it is enriched with active genes and is able to protect such genes from DNA methylation
- DNA methylation prevents H2AZ recruitment
what is the function of H2AZ? H2AX? H3.3?
- H2AZ: gene expression
- H2AX: DNA repair
- H3.3: transcriptional activation
describe H2AX shuffling
- replaces H2A
- associates with DNA regions that need repair
- recruits DRE (DNA repair enzymes) via phosphorylation of a serine
___________ and ___________ localize to chromatin regions where genes are being transcribed
H2AZ and H3.3
describe H2AZ / H3.3 shuffling
- believed to help open chromatin structure
- nucleosomes with H2AX and H3.3 seem less stable than those with H2A and H3
30 nm chromatin fiber
- further compaction of 10 nm beads on a string fiber
- zigzag ribbon / double helix shape
histone _______ promotes tighter chromatin packing
H1
what regions is H1 enriched in?
regions of DNA that are not being transcribed
how/why does histone H1 promote tighter chromatin packing?
- binds linker DNA
- seals two turns on DNA
describe the functions/activities of histone H1
- promotes recruitments of DNA methyltransferases
- inhibits recruitment of histone methyltransferases
H1 recruitment results in tightly packed chromatin associated with…
- methylation of DNA
- demethylated H3 K4
which histone inhibits recruitment of histone H1?
H3.3
what does the LCR (locust control region) do?
- renders adjacent DNA capable of being expressed
- play a role in looping chromatin into structural and functional domains
describe insulator elements (what they do, why they’re important)
- can block the spread of LCR or enhancer activity
- confines LCR activity to particular gene
- prevents spread of closed chromatin structures
how do insulator elements play a key role in looping?
CTCF proteins bind, forming loops
what is the evidence that DNA methylation regulates chromatin structure?
- unmethylated DNA is DNase1-sensitive
- cytidine analog 5-azacytidine incorporates into DNA and promotes gene expression by blocking DNA methylation
what regulates transitions between closed and open chromatin?
DNA methylation recruits inhibitory proteins that promote tight chromatin
what method is used for detecting DNA that is associated with modified histones? how is it done?
- ChIP
- use of antibodies that recognize specific histone modifications
acetylation of lysine is associated with what transcriptional effect?
activation
methylation of lysine is associated with what transcriptional effect?
activation OR repression
methylation of arginine is associated with what transcriptional effect?
activation
ubiquitination of lysine is associated with what transcriptional effect?
activation OR repression
phosphorylation of threonine/serine is associated with what transcriptional effect?
activation
hyperacetylated (many acetyl groups) histones localize to…
active genes that are DNase1 sensitive
hypoacetylated (few or no acetyl groups) histones localize to…
transcriptionally inactive chromatin
what are ways to regulate histone modifications?
- activators can recruit acetylases
- repressors can recruit deacetylases
- acetylases/deacetylases can be regulated
how does acetylation influence chromatin structure?
promotes open chromatin structure by weakening nucleosome associations
what is a way in which acetylases/deacetylases can be regulated?
- MEF2 transcriptional activator associates with HDAC
- HDAC phosphorylation shuttles it to cytoplasm and it can no longer remove the acetyl group
how are histone methylation patterns propagated through cell division?
pre-existing pattern of H3 K9 methylation is inherited due to HP1/HMT activity
chromodomain vs bromodomain
- CD containing proteins induce closed chromatin
- BD containing proteins promote open chromatin
what is a FACT protein?
a protein that regulates elongation of RNA transcript
is histone phosphorylation associated with open or closed chromatin?
open
what does HP1 do? how is it affected by phosphorylation?
- HP1 recruits HMT to methylate H3 K9
- helps to spread heterochromatin, turning off expression
- phosphorylation of H1 blocks its interaction with HP1
what does the histone code state?
DNA transcription is largely regulated by post-translational modifications to histone proteins
how can methylation of H3 promote DNA methylation?
- H3 K9 methylation allows binding by HP1
- HP1 recruits DNA methyltransferases
how can lncRNAs promote the closing of chromatin?
recruit methyltransferases that then methylate DNA and inhibit gene expression
how can lncRNAs promote the opening of chromatin?
the recruited methyltransferase can be sequestered so it can’t methylate the gene, this keeps the structure open
what are the post-transcriptional effects of small regulatory RNAs?
- induce degradation of target RNA
- block RNA translation
what is the transcriptional effect of small regulatory RNAs?
siRNAs can cause tight packing of chromatin
how do siRNAs cause tight packing of chromatin?
- recruit HP1
- recruit HMT
- recruit Dnmt
what is the difference between micrococcal nuclease and DNaseI?
- MN is a single-strand specific secreted glycoprotein that cleaves one strand of DNA as the helix breathes
- DNaseI is a non-specific endonuclease
DNaseI hypersensitive sites are sites that…
- are nucleosome free
- have altered nucleosome structure
chromatin remodeling can be produced by…
(give example)
- proteins capable of displacing nucleosomes or altering their structure
- ex: GAGA factor binding displaces nucleosome so heat shock factor can bind and activate transcription
in what way does/do SWI/SNF alter chromatin structure?
alter in a way that allows other factors to activate transcription
SWI/SNF activity can be modified by…
histone H1
what do pioneer transcription factors do?
bind tightly packed chromatin and open it so that other regulators can interact
describe characteristics of an inactive X chromosome
- CpG dinucleotides are methylated
- 5-azacytidine leads to demethylation and activation of previously inactive genes
- histone codes associated with tightly packed heterochromatin: H3 K9/K27 methylation, reduced histone acetylation, reduced H3 K4 methylation
- enriched with macro H2A
describe characteristics of active X chromosome
- CpG dinucleotides are largely unmethylated
- histone codes associated with lightly packed euchromatin: increased histone acetylation, increased H3 K4 methylation
