quiz #2 Flashcards
why do histone proteins migrate anomalously?
histones don’t migrate because they are very charged
what does B-mercaptoethanol do in SDS gels?
breaks disulfide bonds
epitope tags for protein detection and isolation
GST- binds to glutathione
6HIS- 6 histidines in a row: binds to nickel chelate resin
peptide (epitope) tags: FLAG, myc, HA: bind to specific antibodies
GFP: small protein
TAP: sequentially uses 2 different epitopes
what are epitopes?
tags for protein detection and isolation
antibody binding sites
TAP tags
tandem affinity purification tags
very low background of non-specific interactions
1. purify target protein and interactors using IgG beads, which bind protein A
2. TEV protease removes protein A
3. purify target and interactors a second time using Calmodulin beads
what is ChIP used to detect?
- proteins-DNA interactions, binding
- binding sites and distribution of transcription factors
- gene transcription and polymerase activity
- modifications to histone that influence chromatin structure and gene expression
- nucleosome architecture and regulation of chromosomal maintenance
what can ChIP-exo identify?
protein-DNA interactions with near base pair precision by incorporating exonuclease digestion
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Chromatin Endogenous Cleavage-Seq (ChEC-seq)
- uses micrococcal nuclease (MNase) attached to protein of interest (YFP) via a flexible linker (completly inactive until Ca2+ ions are added)
- introduce YFP-MNase into live cells
- permeabilize cell membrane and add Ca2+
- DNA near (but not bound by) YFP cleaved by MNase
- isolate and sequence small fragments of DNA
omics
Genomics: DNA sequencing
Transcriptomics: RNA-seq
Proteomics: Protein/complex purification and MS
Metabolimics: MS and NMR
what was used in typical large genome projects?
promoters as landmarks
size of human map unit
1,000,000 bp
how many map units in humans is more than all DNA of E. coli
4 map units
high-resolution recombination mapping
landmarks for anchoring sequence information
1cM = 1 Mb DNA
1cm = 1% chance of recombination during meiosis
how identical are humans at sequence level?
99.9
how many SNPs are between any 2 individuals?
3 million
FISH
- fluorescent in-situ hybrid
- cloned DNA with fluorescent dye
- hybridize to denatured metaphase or polytene chromosomes
- chromosome locations
physical maps
based on bp, not recombination
maps of purified pieces of genome (cloned DNA)
clones with large inserts are most useful
overlapping clones are assembled into contigs
Contigs
long continuous stretches of chromosome DNA
BAC sequences
what is the purpose of integrating genetic and physical maps?
to know which chromosome is which
order of markers is the SAME on genetic and physical maps
physical distance (base pairs) is NOT THE SAME as map distance in % recomb
evidence that physical evidence (bp) IS NOT the same as map distance in % recomb
- frequency of recombination can differ 100 fold
- recombination rates are influenced by chromatin structure
- in humans, there are 30,000 recombination hot spots spaced every 50-100 kb
genome sequencing
one consensus sequence per chromosome
<1 error/ 10,000bp
usually 10 independent reads of each ntd
what are the 2 basic genome sequencing strategies
ordered clone sequencing- clones make up a physical map; requires the mapping of each chromosome prior to DNA splitting.
whole genome shotgun sequencing- randomly sequenced clones are assembled; best suited for small (bacterial) genomes; gaps filled by primer walking
ordered clone-by-clone genome sequencing
- overlaps allow fragments to be assembled
- requires the mapping of each chromosome prior to DNA splitting.
first draft of the human genome sequence?
2001
took 13 years and $100 million
how many bp and genes are in genome?
3 billion bp
20,000 genes
how many bp per cell?
60 billion
what % of the human genome codes for enzymes?
1.5%
haplotypes
set of DNA variants tat tend to be inherited together because they are close to each along a single chromosome
(shared group of polymorphisms that are very close together, so stick together)
homolog definition
genes related by descent from a common ancestral DNA sequence
ex: hemoglobin and myoglobin
2 types of homologs
ortholog- gene in different species that evolved from common ancestor. retain the same function during evolution; ancestors and progeny
paralog- genes related by duplication within a genome. May evolve new functions; cousins
C-value paradox
- DNA value per nucleus
- lack of correlation between genome size and developmental, metabolic, or behavioral complexity
what % of genome is transcribed?
more than 80%
what % of genome accounts for introns?
30%
microRNAs and long noncoding RNA
regulate gene expression at epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels
what things have acquired key roles in gene regulation, development, and diseases?
highly repetitive elements, remains of transposable elements, and pseudogenes (inactive genes)
basically, dead genes acquire function
when was the last common ancestor?
90 MYA
what approach is used for comparative genomics?
synteny
* retain the same function during evolution organisms of relatively recent divergence
* show similar content and organization of genes in the same relative positions in the genome (not necessarily in the exact same order)
* one of the most reliable criteria for estabilishing the orthology of genomic regions in different species
how can humans and apes be related?
human chrom. 2 is the result of head-to-head fusion of telomers of chromosomes homologous to chrom 12 and 13 of apes (Robertsonian translocation)
what are hominids?
extint members of the human lineage (not our ancestors, but cousins)
human-specific changes
most proteins have only 1-2 amino acid differences (30% are identical)
changes in conserved noncoding sequences (regulatory regions)
1. brain development (GADD45G)
2. ability to speak and use language (FOXP2)
3. decreased sensitivity to smoke derived toxins (AHR)
what percentage is neandertal geome identical to modern humans?
99.7
what percentage are chimps identical to modern humans?
99.0
most current humans from Europe and Asia but not …….have …….
not Africa
1-3% Neanderthal DNA
Neanderthal genes that show evidence of positive selection
genes that affect keratin function in skin and hair
neanderthal version of some genes involved in immune system function
what could contribute to disease in modern humans?
neanderthal genes
how much Neanderthal genome is present in modern humans?
30%
but some regions appear to be completly lacking
human DNA from –> …….. from —>…….
humans –> Tibetans –> Denisovans
Tibetans
high altitude adaptation caused by introgression of Denisovan-like hypoxia gene, EPAS1
Denisovans
sequences make up 5% of DNA from modern Melanesians, Papuans, and Aboriginals
what makes our brains different from Neanderthal’s?
- 100 mutations that change protein structure are specific to modern humans (only 100 proteins have meaningful aa change)
- TKTL1 active in frontal cortex of humans: glucose metabolism and cell cycle regulation
(if add human version of gene to mice, mice grow more neurons)
(less neurons formed with Neaderthal version of gene than with human) - induce brain organoid fromation in tissue culture, which is an organ made in the lab
how has the human genome continued to adapt and evolve?
- lactase production(evolved independently in Africa, Middle East, and Europe; associated with domestication of cattle, camels
- skin color: fair skin allows vitamin D production under low light; light skin protects against skin cancer in high light
- G6PDH: high levels defend against free radical damage; low levels provide partial protection against malaria
- alpha-amylase: humans have between 1-9 copies of the gene; high numbers in societies with high starch diets (apes and most wild dogs only have 1, Dogs that have been with humans long time picked up additional copies)
what is measured in a northern vs southern vs western
blot?
northern: RNA
southern: DNA
western: proteins
3 requirements for linear chromosome stability and inheritance
telomeres: protect chromosome ends and allow their complete DNA replication
centromeres: facilitate segregation during mitosis and meiosis
origins of replications
telomerase
TER RNA
TERT
telomerase: ribonucleoprotein enzyme (protein + essential RNA)
TER RNA: template for the repeated addition of G-rich telomeric repeats to the chromosome 3’ end
TERT: reverse transcriptase that copies the TER template
how are the ends of chromosomes preserved during DNA replication?
telomerase has its own primer (uses DNA as the primer), uses reverse transcriptase because copies DNA
telomeres promote T-loop structure, which protects DNA ends and blocks DNA damage response
what can telomere shortening lead to?
DNA damage response
cell cycle arrest
deletional recombination
telomere fusion
dangers of telomerase inhibition and activation
inhibition: limited proliferation potential (stem cell disease like aplastic anemia)
activation: unlimited proliferation potential (cancer)
what do telomeropathies cause?
premature telomere shortening –> stem cell disease
muations in core telomerase subunits or accessory factors cause progressive telomere shortening –> failure of stem cells to proliferate
mutations in telomere binding proteins can also cause cell death or genome instability
centromeres
- DNA sequences/regions where sister chromatids adhere to each other most strongly prior to anaphase
- assemble at kinetichore, which is a large protein complex where microtubules of teh spindle apparatus attach to the chromosome (trans-acting factors)
cis-acting factors vs trans-acting factors
cis: affect gene expression on same piece of DNA
trans: diffuse through DNA and affect different genes
chromosome disjunction
cohesins- ring-like proteins that prevent premature separation
separase- cleaves cohesin at anaphase
what is anaphase driven by?
APC and activator Cdc20 –> triggers both activation of separase and also degrades cyclins
less CDK activity –> exit from M phase
by how much do chromosomes compact?
7000 fold
length of DNA wrapped around histones
length of linker DNA
146 ALWAYS
around 50 bp (varies)
nucleosomes
146 bp of DNA (-) wrapped around histone octamers (+)
what makes up nucleosome core?
H2A
H2B
H3
H4
made up of Lys and Arg
core is highly conserved because highly specialized
core has a well-ordered crystalline structure
what does DNA wrapped around nucleosome consist of?
relatively straight 10 bp segments that are connected by bends and the DNA is slightly underwound
A=T vs GC regions in nucleosomes
2 or more A=T at 10 bp spacing will tend to position nucleosomes
GC tracts inhibit nucleosome placement
arrange DNA to where nucleosomes want or dont want to park there
what mediates histone-DNA and histone-histone interactions?
Histone-fold motif
tails of histones
extend from core octamer
unstructured and available for interaction and modification
how many DNA turns around core octamer?
1.65 turns
who has the least neanderthal DNA?
humans from Africa
by how much does chromosome compact from nucleosomes?
6 fold linear compaction
what does linker histone H1 do?
goes at the end and compacts nucleosomal arrays
what do N-terminal tails of histones do?
interact with adjacent nucleosomes compacting chromatin
Whole-genome shotgun sequencing (WGS)
sequence a large number of overlapping DNA fragments in parallel (reads)
uses computers to assemble into largers contigs
primer walking to get scaffolds
