Lecture 16 Flashcards
What does streptomycin interact with?
16S RNA and S12 protein
-both are encoded by the rpsL gene
What can we do with this rpsL gene?
insert it into the pet 21 genome of bacteria
which cells with be ampicillin antibiotic resistant?
cells that contain the plasmid
what regulates protein in pet 21?
lac repressor
how can we put that gene into ecoli?
gibson assembly after we PCR the rpsl-strR gene
epigenetics
changes in genes that are not the result of changes to the DNA sequence
-may let parents send info abt the world to their kids
-all about chromatin structure and DNA methylation
-like putting a word in bold but not changing the letters
How does DNA fit into the nucleus?
it wraps around the histone
each chromosome has it own?
domain/ place in the nucleus
-the domains are dynamic and can change
-chromosomal repositioning/ domain rearrangement
chromosomal DNA hierarchy
-lamina (LAD) where the chromosomes are under the nuclear membrane
-inside the nucleus
-dna loop
-fiber
-chromatin
-dna (smallest)
what are eukaryotic chromosomes?
nucleoprotein complexes
-146 bp of DNA wrapped around histone that has 8 subunits
is the wrapping of histones highly regulates
-yes and so is the spacing between beads
what is a nucleosome bead
8 histone molecules (4 highly conserved) and 146 bp of DNA wrapped around it
-they coil into chromatin fibers that are then coiled into chromosomes
what forms chromatin?
beads on a string,
that chromatin will later be packed by nucleosomes, then the fibers folded into loops
basic nucleosome structure
beads on a string
-seen in interphase
when are the beads on a string visible
when they compact during meitosis
-but then it is harder to find sequences
what do histones have that regulate compaction?
amino acid tails
-they can be covalently modified
-they are also available for chemical modification
-tail modification do not come into contact with DNA
What loosens the chromatin?
-acetylation of the histone tail lysines
-it is also reversible
-the lysine side chain will open when it is acetylated
-remove it= deacetylation (compact again bc the DNA can now wrap around tightly again)
what makes the chromatin more compact?
methylation of the histone tail lysine
-chromatin will be less active bc it is compacted
what can the chromatin state be used for
gene regulation/ expression
-when the gene is wrapped tight around nucleosome the polymerase can express
-wrapped tight= less expression
what recruites histone acetylases to acetylate the histone?
transcriptional activators
-open up the chromosome
what reverses the opening up of the chromosome?
histone deacetylases
chromatin remodeling complexes
move nucleosome aside to reveal TATA/ expose the promoter so transctiption factors and RNA pol 2 can bind tot he DNA
what recruits chromatin remodeling complexes?
transcription activators
what happens when the tails get modified?
they are recognizable by other proteins which will promote assembly or disassembly of histone
can histone modifications effect transcription?
yes, depending on the number of methyl groups present
encode project
use ChIP to map DNA binding proteins in humans
-lets us pinpoint regions of the DNA where the modification is and what type it is (methylated or acetylated)
euchromatin
less packed transcriptionally active DNA
heterochromatin
highly compact and transcriptionally silent
-all methylated
what do mammals do with their X chromosome?
there can only be one active X chromosome so females will put one in “storage”
how does heterochromain stain?
dark bc it is tightly packed
barr body
-transcriptionally inactive bc methylated
-what females do with other X
lyonization
x inactive
what is mosaic and why are females mosaic
-expressing maternal and paternal X genes throughout the body
-females are this bc they have 2 x chromosomes and they each develop independently and activate a different X chromosome
-the choice can not be changed not even by the daughter cells of somatic cells
-everything is euchromatic until the cell decides which genes to keep on
are twins truly identical?
no bc they will have different mosaics which is why they can have different phenotypes
x inactivation
the process of turning off one x chromosome
where does CpG methylation happen?
5 prime CG 3 prime
3 prime GC 5 prime
-prevents transcription factors from binding so this can silence genes
-this is NOT DNA methylation in bacteria when they repair damage
what does methylation interfere with?
DNA binding proteins interacting with DNA bases
-the DNA protein will not recognize the bases so it wont bind
why is folic acid important for pregnancy?
folate is a co factor for methyltransferases
what happens when CpG hypermethylates cells?
-cells dont get the stop dividing signal leading to cancer
-methylation enzymes are over active and demethylases are inactive
-results in the CpG island sequence getting silenced therefore no stop codon/ gene inactivation
imprinting
small deletions of chromosome 15
-can cause prader willi and angelman syndromes
-normal 15 is silences which leads to a homozygous deletion of some genes
-the different outcomes/ illnesses depend on weather the maternal or paternal gene was deleted
paternal 15 deleted
prader willi
-cant stop eating
maternal 15 deleted
angelman
-always happy and jerky movement
methylation is removed where
-in embryonic germ cells that are going to form gametes, all imprints are erased and all genes are active
-the methylations that will be sex specific (maternal or paternal) are later in germ cell development and then transfered to gametes
-the egg will look like mommy somatic cell origianlly looked with each gene active or silenced and sperm will look like daddy cell oriningal did
what happens when there is a deletion on paternal chromosome?
anything active on the maternal chromosome gets expressed like angelman
hunger winter fetuses
higher rate of obesity later in life but they were in utero during the hunger winter
-as adults they had more ares of CpG methyalation and this hypermethylation was near metabolic genes
kids of sick worms from PA14
avoid PA14 even though they have never been exposed to it
-transgeneration memory of PA14 lasts four generation until worms stop avoiding it
training c elegans to avoid pa14
-only need short under 200 nt bacterial RNA to train them
-just exposing them to this RNA and they know to stay away even though they ahve never seen it before
P11 RNA
can train animals to stay away from P14 wihtout ever meeting P14
-give P11 to parents and the kids will avoid it
-parents never met the actual bacteria just the RNA
-has a stem loop complementary to sensory neuron maco-1 in c elegans gene (odor)
-maybe RNAi or miRNA mechanism
RNA dependent RNA polymerase
(RdRP) can amplify RNAi response
-that RNAi response can be passed down
RNAi dicer dcr-1
needed for P11 learning
-transgene will express the dicer in the intestine and suggest intestinal processing of ingested bacterial RNA
