Oct 4 Flashcards
How is nucleosome position determined?
- Use micrococcal nuclease that cleaves DNA between nucleosomes
2.Then use restriction enzymes that cut at certain known points on the strand
3.Run on PAGE - DNA hybridized with labelled (radioactive) DNA (southern blot)
- Get gaps of 150-200, these are adjacent to where the restriction enzymes cut, show where nucleosome was
What are the 4 modifications to histone tails and their effect on the charge of which AA’s?
Lysine gets acetylated (now neutral), methylated (still pos), ubiqutinated.
Ser, Thr, Tyr get phosphorylated (now neg)
What is the histone code?
It is that histones are modified at certain positions, it regulates gene expression
What N-terminal histone modifications are recognized by chromo and bromodomains?
Acetylated lysine recognized by bromodomain, methylated lysine recognized by chromodomain
What histone tails are recognized by SANT domain containing proteins?
Unmofidifed histone tails
How are parental histones and new histones associated with the parental and new DNA during duplication? What does this do for the next generation of DNA?
Parental H3+H4 tetramers are randomly transferred to either parental or new strand, they bind quickly to DNA. Then new H3+H4 tetramers bind to whichever strand does not have one yet.
H2A+H2B dimers are all released into a mixed pool first, then compete with new H2A+H2B for H3+H4 tetramers once they have bound
The new DNA strands now have some parental histone subunits, and they now can see what it was, so it will add the same modifications to the new histone subunit tails to match it. This way the parental strand keeps its function, but the new DNA gets to inherit the same epigenetics
What charge do histone chaperones have? What do they do?
They are neg charged, they escort histones to site of nucleosome assembly
How to determine which nucleotide is modified on histone?
Unmethylated cytosines are deaminated to uracil, methylated ones are protected. Then after PCR the uracil codes for Adenine instead of guanine as its complementary base pair, so can compare to the reference genome and see which ones.
What is high-throughput chromosome conformation capture (HI-C)
It is when DNA is crosslinked, then digested. The frequency of crosslinking determines how close the 2 chromosome strands are, so can link the chromosome structure to the genomic sequence.
