The cell nucleus - chromatin structure

Question 1

electron micrograph

Answer 1

can visualise the nucleus,nuclear membrane, nucleolus, heterochromatin and euchromatin

Question 2

FISH

Answer 2

location of genes and genome in nucleus
you need a probe and target dna - probe is the region your intrested in and the target is the cell your looking at. aka cell nucleus etc

denature probe and target dna
this makes them both single stranded
then add probe to target and they will bind
and when there is a match to probe it will be bound and if a fourescent tag was attatched we can see the target

Question 3

chromsome info

Answer 3

chromosomes occupy different territories in cell nucleus
arms and bands of chromosomes are distinct and variable in shape
chromosomes have preferential positions in interphase nucleus and this correlates with gene density
chromsomes with more expressed genes located in interior

Question 4

genes locations

Answer 4

genes have prefential locations on surface of chromsome territory and can dynamically loop out in response to transcriptional activation
aka genes can be deep in chromosome territory and come out when needed for transcription

Question 5

FISH limitations

Answer 5

sensitivity; how small a single copy genomic region can be visualised
resolution; minimum distance between two genomic regions that can be distinguished as two seperate signals
single cell analysis; a snapshot of what us happening in a cell population.

Question 6

how is chromatin folded to form interphase chromosome ?

Answer 6

2m of DNA wraps around histones to form nucleosomes - beads on a string

Question 7

structure of nucleosome core particle

Answer 7

crystal of nucleosome looked at by x ray crystallography
dna is white in it

Question 8

structural strate of nucleosomes

Answer 8

tetrasome(missing h2a and h2b)

hexasome(h3-h4 plus one h2a-h2b hetero dimer) - are intermediate states during nucleosome assembly and during transcription
variation in handedness of dna fibre
hemisome- half nucleosome
lexosome - transcriptionally poised nucleosome.

Question 9

higher order chromatin packaging

Answer 9

transcriptionally silent highly packaged chromatin base on 30nm fibre
or
transcriptionally active more open chromatin

Question 10

models of 30nm fibre from EM and crystal studies

Answer 10

10nm fibre
solenoid moddel - coiling around central axi 6 nucleosome per turn
zigzag model - two rows of two nucleosomes with linker dna crisscrossing them

Question 11

solenoid model

Answer 11

em chromatin
1976
observations suggest that fibres are formed by winding up of nucleofilaments into helices.

Question 12

chromosome conformation capture 3C

Answer 12

1. crosslink of dna so any two close dna will have cross links
   digestion - digest dna and get left with cross links
   ligation- free ends of dna ligated together,
   ligation products represent chromatin interactions

Question 13

importance of 3c

Answer 13

comparision between genomic regions led on to larger cale expermients such as hi-c which allows comparing one genomic reagion the rest and carry on aka all vs all

Question 14

HI-C method

Answer 14

crosslink DNA
cut with restriction enzyme
fill ends and mark with biotin
ligate
pruify and shear DNA, pull down biotin
sequence using paired ends

Question 15

interpreation of Hi-C data

Answer 15

triangles are TADs
over 2k TADS in human genome