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MCDB 144 Post-Midterm Stuff FOR ASTRID
Terms in this set (105)
How to find specific histone modifications at certain genes?
ChIP, where you treat with formaldehyde and a nuclease to cut the linker DNA
The antibody targets the specific modification of interest
Then you release the DNA from the histone and PCR it with gene of interest-specific primers
What does an "input" control for in an IP?
1. Make sure the protein of interest is there at all
2. Make sure the probes/primers/whatever work
3. Enrichment control
How to directly test protein-protein interactions?
Put the two proteins in another biological system (to prevent the effect of intermediates helping them coimmunoprecipitate) and see if they still coIP without any possible intermediates from original biological system
How does a TAP immunoprecipitation work?
Attach calmodulin to C-term and Protein A to N-term of target protein
Two different IPs where you pull down Protein A, cleave it from the target protein, then pull down again with calmodulin and cleave
Pure AF protein for mass spec
How does paired-end sequencing work?
Like normal massively parallel sequencing but go from both ends, so get double the sequence reads per cycle
What type of DNA do DNA-binding proteins attach to? Why?
Major groove b/c more variable surface so more informative for protein (4 pieces of info in major, 3 in minor)
How do Leucine zippers work? What confers specificity?
DNA-binding proteins that homo- or heterodimerize to get specificity
Helix-loop-helix - differences from -turn-? Function?
DNA-binding protein with larger connecting sequence than turn, also dimerize and help maintain cell fate
Gel Shift Mobility Assay - function? How it works?
Radioactive DNA mixed with cell extract
Run on gel compared to naked DNA, can find sequences bound by proteins because they run larger
DNA Affinity Chromatography - function? How it works?
Find protein that binds specific sequence
Run cell lysate through column that has random DNA sequences, then salt wash to remove non-DNA binding proteins
Run THAT through column with DNA sequence of interest, salt wash to remove all but strongly bound proteins
DNA footprinting - function? How it works?
Find area of DNA bound by protein
Use DNase that cuts everywhere without protein on 5' labeled DNA
Run on gel, find area of no size cuts, that's where protein is bound
Why is eve expressed in stripes?
Reg sequences at TSS, so high Bcd and Kr and low Gt and Hb makes it express
In what order do histone chaperone proteins load core proteins?
H3/H4 tetramer first, then H1/H2
How can DNA be affected to change likelihood of TS initiation (5)?
Alternate histone core proteins
How can transcriptional repressors work (5)?
1. Competitively bind DNA
2. Block surface of activator
3. Block TFII's
4. Recruit CRC to tighten chromatin
5. Recruit HDACs/HMTs to also tighten
What is a bivalent gene?
Has activator and repressor marks on same histone at same time, signals remove one to let the other affect expression
What passes on DNA methylation after replication?
DNMT1, by recognizing hemimethylated DNA
What intermediates are present when removing DNA methylation? Qualities?
5mC oxidized to 5-hydroxy-mC aka 5hmC via TETs, but are NOT inherited
What process are lncRNA's involved in?
How can expression be regulated without changing sequence (3)?
Feedback loops that become self-sustaining
How can proteins affect alternate splicing?
Repressor proteins prevent splicing (leave in alternate exon)
Activator proteins increase splicing (take out alternate exon)
How is alternate splicing involved in sex development?
Sxl in males -> Tra males spliced and nonfunctional -> no Dsx splicing
No Sxl in females -> Tra females not spliced and functional -> Dsx spliced
How can alternate splicing help fix Duchenne's muscular dystrophy?
By tricking splicing machinery to cut out nonsense mutation and make it in-frame, can produce more of protein (including end, which is important)
What helps exports RNA from the nucleus? What types are there?
heterogenous nuclear RiboNuclear Proteins (hnRNPs)
General and Zipcode, which bind mRNA motifs to bring them to certain places (eg bicoid stem-loop)
How can TL effectors interact with zip-code/zip-code binding proteins?
bind stem-loop and affect localization
Difference between siRNA and miRNA?
siRNA not processed by Drosha/DICER, miRNA are
General dsRNA processing in cell
cleaved by DICER (siRNA has loops removed)
loaded into Argonaute to create RISC which cuts it into pieces
Iff extensize match with ssRNA - cleave it
Iff some match with ssRNA - block TL
What does Argonaute and the RITS complex do?
Go to RNA being transcribed like processed dsRNA, repress TS through many mech's (recruit other enzymes)
How are miRNA's processed before DICER?
DGCR8/Drosha cleaves large transcript containing miRNA, then exported to cytoplasm for DICER
What does PIWI do? What do PIWI-binding RNAs do?
Essential for germ cell self-renewal to block transposon activation
Processed in cytoplasm to slightly longer than mi/siRNA (26-30nt)
What is a polyribosome?
Structure of many ribosomes on a single RNA strand spaced ~80nt
2 Examples of aa variations to codon code?
Candida albicans fungus translates codon CUG as S instead of L
MC's translate codon AUA to M instead of I
What is TL recoding? How?
Serine to Selenocystine
Uses its own TL factor to bind GTP
Factor with GTP binds mRNA hairpin loop to add selenocystine
What is TL Frameshifting? How?
Changes protein made by slipping on RNA sequence, eg GaG-Pol by 10% of ribosomes instead of GaG
UUUUU in a row causes ribosome to slip back one nucleotide, change in reading frame creates GaG-Pol
What protein drives nonsense mediated decay and how?
Upf binding EJC's, if STOP before EJC = degraded
When do chaperones start helping with folding?
Before TL ends
How is proinsulin made into insulin?
Ends form disulfide bond, middle is cleaved, connected ends are mature hormone
What do SDS and Mercaptoethanol do?
SDS - length-proportional negative charge
Merc - reduces disulfide bonds to mess up 3/4 structures
How is SRC activated?
Pi removed to loosen binding within protein, then ligand binds SH3 domain and it self-Pi's (elsewhere)
How can p53 involve itself in many different pathways
The thing is COVERED with post-translational modifications
What do probes for unmethylated C's have (after bisulfite seq)?
Many A's, b/c probes recognize T's which used to be unmethylated C's
Germ-plasm theory of inheritance - what is it?
Germ cells are determined by segregated cytoplasmic determinants (like posterior granules in C. elegans)
How are mammal germ cells made?
INDUCED from within the epiblast to form PGC's
How many PGC reprogramming steps are there and what do they do?
2, progressively reduce global methylation
What is a DMR?
Differentially methylated region, causes imprinting on loci
When is DMR methylation removed in PGCs?
Reprogramming step 2
Does methylation in DMRs mean no gene expression?
No, means DIFFERENT gene expression, eg Maternal H19/Igf2 locus unmeth'd so CTCF binds C's and only H19 TS'd, opposite in paternal
What are some ways 5mC's may be removed in PGC's (4)?
1. Direct methyl removal
2. Nucleotide excision repair
3. Base excision repair
4. TET's do their 5hmC thing
How are DMR's remethylated in germ cells?
Dnmt3L and 3a cofactor
What defines a "malignant" carcinoma?
Breaking out of epithelium via basement membrane
What is a neoplasia?
A nonmalignant carcinoma
What does a pap smear do?
Test cervical epithelium for cancer - lower differentiation, more division, and high nuclear size
How to detect genetic abnormalities in cancer on different scales?
Real big - karyotype
Sorta big - FISH
Small - Microarray
How can epigenetic changes lead to cancer?
Accidental hypermethylation/heterochromatin formation of tumor suppressors
How are different tumor-causing mutations classified and arranged to form a malignancy?
Initiators - one only, needs to happen first
Promotors - many of them need to be clustered some time after Initiator mutation
How can viral insertion cause cancer (3)?
Activate protoncogenes via LTR
Truncate tumor suppressors (eg Rou's Sarcoma where viral version's C-terminal of Src gone so it's constitutively active)
How to ID cancer genes?
Treat fibroblasts with mutagens, find cancer multilayers, genotype
Why is loss of telomeres bad?
Lose important DNA
Get daughter cell without it after replication, and when THAT cell tries to replicate, the sister chromatids will fuse at the ends and mitosis breaks them randomly
What is the common cause of Chronic Myelogenous leukemia and how is it treated?
Philadelphia chromosome that fuses Bcr-Abl, inhibitor of fusion protein "Gleevec" blocks ATP-binding site
What cells have telomerase activity?
Embryonic stem cells
What is a stem cell niche?
Area where the adult stem cells reside, special signaling areas to maintain stem cell population
What does the Notch pathway do in all tissues?
Help with their development
Where are hair stem cells located?
In the bulge, which is the only part of the shaft that doesn't degenerate in the hair cycle
How does new hair shaft grow?
Bulb goes from quiescent to dividing
Which transcription factors are affected by tetracycline and what do they do? How were they used in the H2B-GFP mouse?
rtTa: induced by tetracycline
tTa: repressed by tetracycline
tTa under Keratin 5 promotor, H2B-GFP under tTa-controlled promotor
Cells that still had GFP after 4 weeks were quiescent, found to be bulge cells
How to test multipotency of cell type?
Isolate and xenograft to nude mice, see if they develop into many different cell types
What maintains hair bulge cell identity? What activates hair bulge cells to proliferate?
B-catenin via Wnt signaling maintains ID, consitutively stable = precocious bulge cell activation and conditional KO destroys stem cell identity
Ki67 activates proliferation, without Wnt - overactive and becomes normal proliferation
How did Venter get a whole chromosome into a bacteria? And destroy the host genome?
Cut it into pieces with overhangs to be HR'd together
RE's that only recognize host genome
How can you transfect DNA into a cell?
Lipoooooo - surround that bitch with a liposome
electroporate - make holes and hope it goes through
How to make self-inactivating viruses?
Nonfunctional viral production proteins, eg GAG, pol, Env
ZFNs, how do they work?
Sequence contains multiple Zinc Finger motifs into an array, which each bind 3 specific bases
Add nuclease sequence eg Fok1 or add TF to change expression
TALENs, how do they work?
Sequence contains multiple TALE repeats where 12/13th base confers substrate base specificity
Then add nuclease or TF sequence
What do the PAM sequence and tracrRNA do in CRISPR?
PAM (NGG or NCC)- nuclease cuts 3-4bp away
tracrRNA - holds Cas9 to gRNA
How to get site-specific integration with transgenic?
Use plasmid that effects HR using +/- selectors and homologous arms, then select for integrated cells and inject into embryo
How to make time-controlled tissue-specific transgenic?
Cre-ER, connected to estrogen receptor so only goes into nucleus and removes floxed site when treated with tamoxifin
Why was cell fusion important in stem cell biology?
Showed cells can be reprogrammed, and that's crazy
How to find pluripotency-related genes?
Diff expression between pluripotent and somatic cells
Find biggest differences, focus on TFs
Clone out cDNA, put in retrovirus, overexpress in somatic cells
How to check for contamination in hiPSC population?
DNA fingerprinting with small nucleotide repeats, different repeat number = different donor
How does pluripotent reprogramming change genomes?
Doesn't affect karyotype but does cause sub-karyotypic changes like repeats
What factors affect hiPSC similarity to hESCs (4)?
Passage number (later is closer)
4 factor vs polycistronic induction
Target cell type
What is IRES and how does it help with iPSC creation? What factors help them work?
Internal Ribosome Entry Site, mRNA seq's that form hairpin recognized by ITAF's to restart TL
Allows for polycistronic mRNA TL
2A sequences allow for ribosomal skipping, release of nascent peptide, and restart (on proline)
How to cause iPSC reprogramming without genome integration?
Plasmid transfections, but do not cause strong enough change unless integrated
Adenoviruses had same problem
How are nucleosomes remodeled?
ATP-dependent chromatin remodeling complex facilitates nucleosome sliding
How are next-gen sequencing results actually measured?
Fluorescence, where added nucleotides have specific colors which are cleaved off before next nucleotide is added
What are the functions of the linker histone?
Wrap and protect gDNA
Condense into 30nm fiber
Change exit path of DNA
What does the clamp loader do in the clamp-slider complex? What does it NOT do?
Loads clamp onto DNA, does not hold it to DNA pol
How to recognize specific histone modifications?
What are the functions of:
H2AX - DNA repair/recombination
H2AZ - gene expression, chromosome segregation
What area of DNA are ORCs normally found?
Euchromatin, to make it easier to bind factors than tightly packed heterochromatin
Two nucleotide analogs that can be identified upon insertion?
BrdU (antibody) and radioactive Thymine (silver)
5 broad stages of expression regulation?
1. TS control
2. RNA processing control
3. RNA transport/localization control
4. TL control
5. Protein activity control
How is secondary structure important in DNA binding proteins?
Specialized to fit into major groove
Using Zn2+, Zinc Finger Motifs coordinate secondary structure to better bind DNA specifically
How can telomerase make more telomeres?
Built in RNA part that acts as a primer
Four functions of RNA polII CTD?
1. Ser5P recruits 5' capping machinery
2. Scaffold for CstF and CPSF for 3' polyadenylation
3. Holds exonucleases to cleave finished transcript
4. Holds splicing machinery to recruit U1 and U2AF
What is the difference between small nuclear RNAs and small nuclear Ribonucleoproteins?
snRNAs recognize the splice sites on mRNA.
snRNPs are protein-RNA complexes containing snRNAs which form the splicosome.
What does dimerization do to DNA-binding proteins?
Increase their activity or decrease it, depending on the type.
3 techniques to ID DNA-binding proteins?
Gel Mobility Shift Assay
How do trans-eQTLs get all the way to target DNA?
Mediator complex brings them close together
How to assay affect of gene regulatory sequences?
Create transgene with reg sequence, promotor, and reporter gene
What part of mRNA controls localization?
Difference between tumor initiators and promoters?
Initiator - causes change to DNA (radiation, ROS, viral insertion)
Promoter - Doesn't change DNA sequence but changes expression (altered methylation, overactive expression from exogenous factors, etc)
What does APC do?
Part of APC/GSK complex, which degrades B-catenin unless Wnt signaling happens. No APC = uncontrolled Wnt signaling = cancer formation
How to demonstrate reprogrammed iPSCs are pluripotent?
Culture them - make three germ layers?
Inject into nude mouse - make teratoma?
Look at expression of pluripotency markers
How can Base Excision Repair remove DNA methylation?
Demethylation and deamination (by AID) of 5mC to T, then Base excision repair
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