Lecture 2 - Eukaryotic Chromosome

Question 1

Approx ? Of DNA in a single human cell?

Answer 1

1.8m

Question 2

What is the compaction needed just to fit into the nucleus is approx how many times?

Answer 2

180,000

Question 3

What is the role of packaging DNA?

Answer 3

Protects from damage and to regulate DNA metabolism

Question 4

Where can evidence of DNA packaging be found?

Answer 4

Electron microscopy DNA in non-diving cells forms chromatin: compaction DNA during mitosis forms chromosomes

Question 5

What is the chromatin structure?

Answer 5

Chromatin has a fundamental subunit structure - present in virtually all eukaryotic organisms Several levels of organisation are found to form chromosomes Levels vary depending on whether a cell is in interphase or undergoing mitosis or meiosis

Question 6

What is nucleosome?

Answer 6

A repeating unit of chromosome

Question 7

Each nucleoside compromises .. molecules each of … core histones

Answer 7

2 molecules 4 core histones

Question 8

Each core histone dimer has … DNA binding surface that organise … DNA turns

Answer 8

6 core histones 3 DNA turns

Question 9

The histone octamer organises … of DNA in … helical turn of DNA

Answer 9

145bp and 1 3/4

Question 10

How much DNA is packaged in a disc of 6x11nm?

Answer 10

48nm

Question 11

How are nucleosomes prepared?

Answer 11

Individual nucleosomes may be prepared by limited digestion of chromatin with micrococcal nuclease followed by separation of the particles on sucrose gradients

Question 12

Mono nucleosome

Answer 12

200bp

Question 13

Trimmed nucleosome

Answer 13

165 bp

Question 14

Core particles

Answer 14

146 bp

Question 15

What are histones?

Answer 15

Highly conserved small basic proteins

Question 16

How are histone readily solubilised from chromatin by dilute acid ?

Answer 16

Arctic acid or HCL Rich in positively charged arginine and lysine

Question 17

What are species of histones ?

Answer 17

There are 5 species designated H1 to H4 H5 is a type of H1 Histone H2A, H2B, H3 and H4 form
Various aggregates

Question 18

A particular aggregate forms the core structure of the nucleosome in the .. fibril?

Answer 18

11 fibril

Question 19

Which histone is not present in the nucleosome?

Answer 19

H1 (H5)

Question 20

Which histones have variable regions?

Answer 20

H1 (2), H2A (2), H2B (1)

Question 21

Which histones preferentially aggregate to form tetramers ?

Answer 21

H3 and H4 Tetramers contain 2 molecules of each histone

Question 22

Which histones also aggregate but in stages?

Answer 22

H2A and H2B

Question 23

What does the size of aggregate depend on?

Answer 23

Initial concentration of 2 histones

Question 24

What are the 5 different inteactions of histones with DNA ?

Answer 24

1. Helix dipoles from alpha-helices in H2B, H3 and H4 cause a net positive charge to accumulate at point of interaction with negatively charged phosphates of DNA 2. h-bonds between DNA backbone and the amjne group on the main chain of histone proteins 3. Non polar interaction between histones and deoxyribose on DNA 4. Salt link and hydrogen bond between side chains of basic amino acids lysine and arginine and phosphate oxygen on DNA 5. Non-specific minor groove insertions of the H3 and H2B N terminal tails into 2 minor grooves each on the DNA

Question 25

What removes histones?

Answer 25

2M Nacl

Question 26

What structure does 30 fibre have?

Answer 26

Solenoid

Question 27

What is 30nm solenoid fibre AkA?

Answer 27

Heterochromatin

Question 28

What is 11nm beads on string AKA?

Answer 28

Euchromatin

Question 29

What are the features of Euchromatin?

Answer 29

Extended structure Active genes

Question 30

What are the features of heterochromatin?

Answer 30

Highly condensed Inactive genes

Question 31

What is the first level of organisation?

Answer 31

11nm fibril

Question 32

In the 11nm fibril, how many number of np of ds DNA wrapped...around on the outside of a flat cyclindridal disc of small BASIC protein?

Answer 32

147 bp and 1.67 turns

Question 33

What is spacer or linker DNA?

Answer 33

Each nucleosome is joined to its neighbour by the DNA which runs the length of the chromosome The short length of DNA joining any two nucleosomes is termed spacer or linker

Question 34

What is the length of spacer DNA?

Answer 34

Around 50bp

Question 35

What is the packing ration of the 11nm fibril?

Answer 35

6 A 6 fold reduction in total length

Question 36

What comprises of the 11nm fibril?

Answer 36

Euchromatin and heterochromatin

Question 37

When is the 11nm present ?

Answer 37

Interface Meiotic/mitotic division

Question 38

What is the transcriptonally active form of chromatin?

Answer 38

11nm fibril

Question 39

How can chromatin be assembled into 11nm fibril?

Answer 39

In vitro on mixing, in low salt, DNA and 4 pairs of histones (H2A,H2B, H3, H4)

Question 40

What is the 2nd level of organisation?

Answer 40

30nm fibril

Question 41

What does the 30nm fibril form?

Answer 41

Left handed solenoid helix

Question 42

How many nucleosomes per turn in the left handed solenoid helix of the 30nm fibril?

Answer 42

6

Question 43

What is the packing ration of the 30nm fibril?

Answer 43

400

Question 44

What is required for the condensation in the 30nm fibril?

Answer 44

Histone H1

Question 45

When is the 30nm fibril present?

Answer 45

Interphase | Mitotic/meiotic division

Question 46

When is the 30nm fibril inactive?

Answer 46

RNA synthesis (transcription)

Question 47

How can chromatin be assembled into a 30nm fibril?

Answer 47

High salt solution mixing DNA and ALL histone species, including histone H1

Question 48

What is the 3rd level of organisation?

Answer 48

Loops, coils and rosettes

Question 49

What is loops?

Answer 49

75kb of DNA

Question 50

What is rosette?

Answer 50

Around 6 loops

Question 51

What is coils?

Answer 51

Around 30 rosettes

Question 52

Looping and cooling the chromatin results in a reduction in total length to: ... in heterochromatin ?

Answer 52

Ca 10,000

Question 53

What are probably involved in condensation?

Answer 53

Specific packaging proteins

Question 54

What are not present in Euchromatin?

Answer 54

Rosettes and coils

Question 55

What is the 4th level of organisation?

Answer 55

Chromatids

Question 56

When is chromatin maximally condensed?

Answer 56

Mitosis

Question 57

What is the packing ratio in mitosis?

Answer 57

>10,000

Question 58

What is the length of chromosomal DNA at mitosis?

Answer 58

~5 micrometer

Question 59

In a single human chromosome DNA ranges in size from ... (smallest) to ... (largest)

Answer 59

1) 50 x 10^6 2) 250 x 10^6 bp

Question 60

What is the full length of DNA in a chromosome of stretched?

Answer 60

1.7cm - 8.5cm

Question 61

What is deproteination?

Answer 61

Chromatin DNA make loop to and from the central scaffold

Question 62

What is the scaffold?

Answer 62

A network of proteins that cross-tie DNA loops

Question 63

Topoisomerase II and Protein SC2 (XCAP C/E)

Answer 63

Major component Both proteins are required for chromosome assembly

Question 64

What is SAR?

Answer 64

Scaffold attachment regions Protein bind to SAR in chromosomal DNA

Question 65

What is SAR?

Answer 65

Highly AT-rich DNA ~ 1 - 2 kB in size

Question 66

What is the role of SAR?

Answer 66

SAR define the bases of the DNA loop Participate in chromosome assembly and structural maintenance