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Cmmb 411 > Topic 4 > Flashcards

Topic 4 Flashcards

1
Q

What is the difference in prokaryotes and eukaryotes abou their dna

And where they are

A

Prokaryotes DNA is inside a nucleoid, less compact

Eukaryotes inside nucleus, very compact

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2
Q

What is special about prokaryotes and eukaryotes chromosome

What I special abiut the prolaryotes

A

Prokaryotes have a single circular chromosome

Eukaryotes have multiple linear chromosomes

They have a plasmid which is accessory content

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3
Q

What is the ploidy of prokaryotes

Eukaryotes

A

Haploid

Diploid or haploid

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4
Q

What happens to the gene as organisms get more complex

A

More complex organism

Highe number of choromosme

More copies of chromosomes

The complexity of the genetic content is increase

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5
Q

What is the genome size definition

Why is it important

A

The total amount of the genetic content in a haploid organism (one copy of the genome)

So if it’s listed as 3200 for humans, it’s actually the haploid of human not diploid

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6
Q

What is the exception to saying that a more complex organisms has a higher genome size

A

Doesn’t work all the time Because some organisms of similar complexity have different genome sizes

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7
Q

What is gene density

A

The average number of genes per mega base (Mb) of genomic DNA

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8
Q

What types of organisms have lower gene density and why

A

More complex organisms because they have a bigger genes size and more dna between genes (intergenic sequences)

Inverse relationship with gene density

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9
Q

What are intergenic sequences

A

DNA between genes

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10
Q

When genome size increases in more complex organisms what is actually increasing

A

More introns

More repetitive DNA

Longer intergenic sequences

NOT JUST GENE NUMBER

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11
Q

What two categories is the human genome split into

A

Intergenic DNA (> 60% of the content)

Genes and gene related sequences (<40%)

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12
Q

What’s included in intergenic DNA

A

Other intergenic regions

Genome wide repeats (majority of the genome is repeats)

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13
Q

What is example of Genome wide repeats

A

Transposons and other mobile DNA’s

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14
Q

What included in Other intergenic regions in intergenic DNA

A

Unique DNA (regulatory regions, microRNA)

Microsatellites (simple repeats, AC100)

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15
Q

What is genes and gene related sequences split into

A

Genes (48 Mb)

Related sequences

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16
Q

What is included in related sequences in gene and gene related sequences

A

Introns and UTR

Gene fragments

Psuedogenes

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17
Q

What are introns and UTRS

A

UTR are untranslated regions like the poly A tail

Introns include microRNA (non coding functional rna)

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18
Q

What do intergenic sequences encode

A

DNA sequences that are Transcribed into Functional rna

But not proteins because that RNA doesn’t get translated

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19
Q

What are functional non coding RNA examples

A

MicroRNA

rRNA

TRNA

snRNA (small nuclear RNA, spliceosome)

Important for cellular function but don’t get translated to proteins

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20
Q

How do you find the percent of genome that’s actually coding

A

Human genome is 3200 Mb

Genes are 48 Mb

48/3200 = 1.5 % actually coding

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21
Q

Which genes related sequences are non function

A

Genes fragments

Psudogenes

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22
Q

What are the impotent parts of a chromosome

A

Kinetochore

Centromeres

Telomeres

Origins of replication

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23
Q

What is the kinetochore

A

A elaborate protein complex that is on the centromeres to interact with spindle surging segregation in celldvision

Trilaminar: three layered structure

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24
Q

What are the centromeres

How many per chromosome

A

DNA sequences that are needed for the kinetichore complex to from

This is the first place we see construction of a chromosome

1 centromere per chromosome

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25
Q

What are telomeres and sequence

How many per chromosome

A

They are TG rich repeats that cal the ends of the chromosome : (TTAGGG)n

Protect from damage and loss

2 pairs per chromosome

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26
Q

What are the origins of replication

A

Sites on the chromsome where DNA replication machinery assembled and starts replication

Many origins per chromosome

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27
Q

What happens if chromosome has one

No

2

Centromeres

A

1: equal segregation (one per cell)

None: spindle fibres can’t attach , chromatids can’t move in certain direction (random segregation)

2: chromosomes break, not equal segregation bc of more than one centromere

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28
Q

Why are telomeres so important

A

They prevent recombination and degradation of DNA (by distinguishing the chromosome ends from the DNA breakage sites)

They act as a specialized origin of replication for replicating the ends of the chromosomes (via telomerase)

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29
Q

What is telomerase

A

A reverse transcriptase

They reversely replicate the telomeres sequences By using the original telomere sequence at a template

To put in the new chromosome

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30
Q

What are the stages of mitosis

A

Interphase (G1,S,G2)

Mitosis (M)

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31
Q

In what pint of the cell cycle does dna condense

A

Between G2 and M

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32
Q

What what pint in the cell cycle does DNA replication happen

A

Between G1 and S

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33
Q

What parts of the cell cycle mark structural changes of the chromosome

A

DNA replication

DNA condense

Chromosome segregation (in M phase)

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34
Q

What are the two checkpoints in cell cycle

A

G1 to S: to check if DNA actually replicated

G2 to M: to check if chromosomes properly aligned before segregation takes place

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35
Q

Interphase is ____

M phase is ____

A

Replication

Segregation

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36
Q

What is the purpose of the GAP phase (G) in the cell cycle

A

To prepare for the next phase of the cell cycle

To check for the completion of the previous phase (checkpoints)

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37
Q

What are the key events in S phase

What holds the duplicated chromsomes together

A

DNA replication starts at the origins and spreads in both directions (many origin sites going up and down)

Each chromosome of a duplicated pair is called a chromatid (both in one pair called sister chromatids)

Cohesion (forms rings to hold them together, important for chromosomal integrity)

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38
Q

What has to happens for the replicates dna to get segregated (metaphase to anaphase)

What did this mean

A

The cohesin has to be removed through degredation

Protein synthesis and degradation it’s important for mitosis (making and degrading cohesin)

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39
Q

What is bivalent attachement

A

Both spindles are attached to the kinetochore

This allows proper segregation

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40
Q

What is the MTOC/centosomes

A

The things that the spindles that attach to the kinetochore on each chromsome come from

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41
Q

In what phase of the cell cycle are the chromosome less compact

When are they most compact

What does thsi mean

A

Interphase (G1,S,G2)

During mitosis, chromsome condensation allows segregation

From interphase to M PHASE there is a drastic change in chromosome topology

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42
Q

What phase is most important to shape the chromosome structure

A

Early prophase

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43
Q

Why does dna loop

A

It does this as a way to package the dna

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44
Q

What are the SMC proteins

A

Structural maintence of chromosome proteins

Cohesin and condensin

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45
Q

What does condensin do

What happens if degraded

A

Condenses the looping dna structure to keep it more compact for mitosis

If degraded the dna goes from compact to loose (to go back to g1 phase)

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46
Q

In meiosis 1 and 2 what type of spindle attachment are there and what do they do

A

Meiosis 1: Monovalent , changes chromosme number from diploid to haploid

Meiosis 2: bivalent, changes chromatin number (sparsrated into 4 cell not two) not chromosome number (because still haploid)

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47
Q

In interphase what are the forms of the chromatin structure (sizes)

In m phase what are the forms of the chromatin structure

A

Two forms: 10nm (beads on a string) and 30 nm fiber (more compact)

M phase: maximum condensation

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48
Q

Why does dna have to be compact

A

The diameter of the nucleus is 10-15 micrometers

In a diploid cell the dna is 2 m

So this long dna has to be compacted 1000 to 10,000 times to fit in the nucleus

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49
Q

How is dna compaction achieved

A

By forming DNA complexes with proteins (ie. chromatin) to from chromosomes

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50
Q

How is a nucleosome formed

A

The dna is negatively supercoiled into the histone core

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51
Q

What is the advantage of DNA packaging

A

The histones protect the DNA from damage

Allows for proper segregation during cell division (because stops entanglement)

52
Q

What is the disadvantage of DNA packaging

A

the DNA needs to be unwrapped for transcription to happen

So being packages reduces the accessibility of the cellular machinery that’s needs for cell function (repair, recombination, replication,transcription)

53
Q

What type of structure is a nucleosome

A

A chromatin structure (dna plus protein)

54
Q

What are nucleosomes made of

How many times does dna wrap around it

How much commotion happens due to the nucleosome

A

8 histone protiens with dna wrapped 2x around the histone core

6x

55
Q

What connects the multiple nucleosomes

A

The linker dna between nucleosomes

56
Q

How did they discover the nucleosome

A

Treated the chromsome with a MNase

Ran gel electrophoresis

Since histone protects DNA from nuclease activity

If digestion is complete (no linker dna) full digestion shows only one 147 bp band
This band is the DNA wrapped around the histone

If incomplete, not just one histone since linker dna not cleaved

So bands at 200 400 600 etc. show since one histone is 200

57
Q

What is MNase

A

Micrococcal nuclease

It’s a sequence non specific nucelase

Quickly Cleaves protein free dna but cleaves protein accosiated dna badly

58
Q

What did the discovery of nucleosome experiment tell us

A

The nucleosome needs at least 147 bo of dna to form

59
Q

How long is the linker dna

A

20-60bp

60
Q

What amino acids are rich in proteins

A

Highly basic lysine and arg

Positively charged amino acids

61
Q

How did the lysine and arg residues affect how we see the histone MW on SDS PAGE

A

Since positively charged, the lysine and arg make it travel slower on ads page (toward the negative)

This makes the MW seem bigger than we thought

Instead of 10-20 kDa like we thought it shows as 30 kDa

62
Q

What is special about the n and c term of histone proteins

In between

A

They have long n term and short c term

In between is the histone fold domain which is made up of alpha helices

63
Q

What are the diff types of histone subunits that make a histone core for DNA to wrap around (and make nucelosome)

A

H2A H2B H3 H4

64
Q

How is a nucleosome assembled

A

H3 and H4 form a tetramer (since two of each)

H2A and H2B make a heterodimer (only one of each and diff so hetero)

The H3H4 tetramer can bind to the middle and ends of the dna in the inside to make them bent into a circular structure

Then the two of the H2A H2B dimer add onto the tetramer on both sides of the tetramer (so not on the inside)

The amino tails stick outward

65
Q

In a histone what makes it an octamer

A

Two dimers of H2A and H2B

One tetramer of H3 H4

Total 8

66
Q

How does the DNA contact the histones (H3H4)

What is important about this

A

Hydrogen bonding near the minor grooves of the dna, also the arg lys of histone is postive and the backbone is negative

The contacts are sequence independent (no specific sequence needed to bind to histones)

The hydrogens bonds allow for the energy to being the dna into circle to wrap around

67
Q

What is special about the n term tails of histones

A

They are not important for the bending or binding of dna to the histone octamer

They are sensitive to proteases meaning they are not tightly folded in comparison to the histone octamer

They can be modified to regulate the function of the nucleosome

68
Q

What do the histone tail help with

A

The tails hide the wrapping of dna to the histone core

And the negative supercoil directionality of the dna

69
Q

How do the histone tails protrude out

A

Some tails go between the gaps in the double helices (H3 H2B)

Some go above or below the dna (H4 H2A)

70
Q

How can the negative supercoiled dna in eukaryotes be relaxed

What is good about being negative supercoil

A

By topoisomerase

This favours DNA unwinding to allow access to dna during replication transcription and recombination

71
Q

What are the two topoisomerase sin prokaryotes and what do they do

A

Gyrase: introduces negative supercoils using atp

Reverse gyrase: keeps genome positive supercoil using atp (used by thermophiles)

72
Q

What is heterochromatin and euchromatin

What is the staining

A

Heterochromatin: condensed, dense staining, higher order of nucleosomal dna assembly, closer to nuclear envelope

Euchromatin: open structure, poor staining, less organized nucelosomal dna assembly , centre of nucleus

73
Q

How did they see the nuclear scaffold/matrix region of the nucleus

A

They treated the cell with high salt, washed away the proteins

Found there were still proteins in the nucleus that are forming scaffolds close to the nuclear envelope where we see heterochromatin

74
Q

What causes further condensation of dna onto histones

What does it do and what is special about it

A

The extra histone: linker histone (H1)

It interacts with the linker DNA to turn the 10nm to 30nm fibre, meaning is also protects the 20-60bp linker region DNA from MNase digestion

75
Q

Why would we use LYS ARG rich histones for forming chromatin structures

A

Because they interact tight with negative charged DNA backbone

76
Q

What forms the 30nm fibre

A

Nucleosomes plus the H1 linker histone

77
Q

What are the two form of the 30nm fibre

A

The solenoid (hollow inside)

Zigzag: the linker region crosses between histones so no hollow inside

78
Q

If the linker region is short, what form of packaging would the 30 nm fibre take

A

Solenoid

Longer is zigzag

79
Q

When the Christin turn to 30 nm. (Getting more compact) what happens

A

The DNA is less accessible to the DNA dependent enzymes (like polymerases)

This causes less transcription/replication, so less expression

80
Q

In the 30 nm structure, what is required to stabilize it

A

The histone tails

They interact with negative DNA on neighbouring nucleosomes since postive charge

So through charge neutralization they allow compaction

81
Q

How much fold compaction does the 30 nm fibre give

A

From 6x (bead on string) to 40 x compaction (30nm)

Not enough compaction

82
Q

How is the rope like structure of the 30 nm fibre made

How big are the loops

A

Onto a chromosome scaffold there are multiple chromatin loops (40-90kb)

83
Q

What are the nuclear scaffold proteins that form the 30nm rope loops

A

Topo II

SMC

84
Q

What does topo 11 scaffold protein do to form the 30nm loops in a scaffold

A

Holds the DNA at the base of the loops and makes sure they are topologically isolated from each other

85
Q

What do SMC scaffold proteins do to form the 30nm loops in a scaffold

A

Condense and hold the sister chromatids after chromosome duplication into the nuclear scaffold

Provides the foundation for interactions between the nuclear scaffold and the chromosomal DNA

86
Q

Exaplin the process of forming nuclear scaffold with the 30nm chromatin

How is it undone

A

During interphase cohesin makes and stabilizes the DNA loops into a more compact structure

Topo 11 comes and bind to the dna to introduce knots by cutting and threading stands through each other (can either make or remove the dna entanglements, right now it’s making them)

The condensin can use dna loop extrusion activity to constrict the the inter and intra molecular interlinks (to untangle them)

87
Q

What are the three ways during formation of chromatin scaffold that Topo two can make knots

A

Within one loop

Knots with Nearby loops :

intra (loops from the same dna strand

Inter (loops from diffent dna strands)

88
Q

What are the histone variants and why are they here

A

H2A.X

CENP-A

Pack the dna

89
Q

What is H2A.X

A

H2A VARIANT, it’s phosphorylated

Its found wherever there’s a double stranded DNA break and is recognized by the DNA repair enzymes

Good indicator of meiosis recombination (which breaks the dna recombine it)

Used for antibodies to recognize the H2A.X to show breakage of DNA that needs repair

90
Q

What is CENP-A used for

How much similarity does it have to H3

A

Replaces the H3 , 40-50% similarity to H3

Found in nucleosome that have bound dna with a centromere (found in region of the dna that are centromeric)

Act as a binding site for the kinetochore to allow proper serration and stability

91
Q

What is used for sperm in mammals packaging

Why

What richness does it have

A

Sperm nucleus is smaller than somatic nucleus

Use protamines instead of histones, arg rich, packs the dna tightly to for the haploid genome into the sperm nucleus

92
Q

How could you distinguish between the sperm and somatic dna on a agarose gel restriction digest

A

See 147 base pairs for somatic dna (full digest)

Sperm cells have no protection from his room since no histones, so you see a smear of a bunch of bands

93
Q

What do DNA binding proteins (rna pol, dna pol) prefer to interact with

A

Histone free dna

Want no nucleosome structure

94
Q

Since dna binding proteins prefers to. Interact with histone free dna what does this mean

A

The histone octamer hinder dna accessibility

So we need relaxed dna for gene expression

95
Q

What regulates the accessibility of chromatin

A

The dynamic nature of the octamer binding to dna

The chromatin remodeling complex changing the binding of histones to the dna to make some regions more open and some more compact

Modification of the N term histone tails

Or all other these this together

96
Q

What region of the dna on histones is most accessible

A

The regions closest to the entry and exit points of binding to the histone

Ones just coming into nucleosome structure or just leaving the structure

Not in between because wrapped around the histone

97
Q

How can chromatin/nucleosome remodelling complex change the movement of the nucleosome

What does it use to do this

A

NRC (nucleosome remodeling complexes)

Use atp to

Slide the nuceleosomes away from the accessible binding sites

Can eject nucleosome fully out of the region that needs to be transcribed

The histone dimer (H2A H2B) get exchange with other dimer by the remodeling complex. Ex. H2A to H2A.X

98
Q

How do the remodeling complex use nucleosome positioning to allow dna acceibility

A

The remodeling proteins bind to the dna and make it so that the sequence between them is less that 147 BP

This causes no nucleosome there since nucleosome need at least 147 BP to form

Makes a nucleosome free region and accessible DNA.

99
Q

How do the remodeling complex use nucleosome positioning to stop dna acceibility

A

Bind to the accessible binding site and recruited the histone subunit

makes it so that a nucleosome forms there

100
Q

Nucelosome prefer to bind what type of DNA

A

Bent

101
Q

What nucleotides in the minor groove actually contact the histone and why

A

AT rich

This is because it’s more flexible (since less h bonding) and can be bent more easily for DNA to bind to histones

Also the electrostatic charge of AT is more negative

102
Q

Does protamines maintain the nucelosome structure

A

No

103
Q

More open dna structure means what in terms of nucleosomes

A

Less nucelosome formation

104
Q

How do researches see the position of nuceleosomes in dna

A

Isolate the nuclei

Treat with MNase to make double stranded DNA breaks between nucelosomes

Treat with a restriction enzyme to make defined end points for all fragments

Run this on agarose gel, then make a replicate on nitrocellulose membrane

Then they design probes for the regions of dna they’re interested in and do southern blot

Found that if the nuceleosomes are randomly placed in the chromsome, cut is done randomly, see a smear of bands

If the nucleosomes are in specific positions next to the sequence they’re interested in, see 160-200 bp bands of the nuceleosomes

105
Q

When doing the experiment to see nucleosome position, what do they mark the probe bind to

A

Usually interested in a specific regulatory sequence of the dna

106
Q

How can the hormone tail be modified

A

Acetylation

Phosphorylation

Methylation

Ubiquitination

107
Q

Which amino acid residues get commonly phosphorylated

A

Serine, tyrosine, threonine

108
Q

What aA get methylated or acetylated or ubiquinated

A

Lysine

109
Q

What does modification of histone tails do

A

Affect protein association which affects the nucelosome function and gene expression

110
Q

What is the histone code

A

The multiple modifications of the histone tail that change the function

111
Q

What does histone acetylation do

What protein domain does it recruit

A

HAT adds acetyl group

Reduces positive charge of tail, Opens the dna, Increases gene expression

Recruited the bromodomain

112
Q

What does histone methylation do

A

Makes more postive charge, bind tighter to dna, less gene expression

Recruit chromodomain

113
Q

What proteins interact with unmodified histone tails

A

Proteins that have the SANT domain

114
Q

In what way do modifications work together to change gene expression

A

In a combinatorial way

Combination of these modifications can change chromatin structure and expression

115
Q

What other way can chromatin structure be regulated

A

Competition between histone and specific dna binding protiens

116
Q

What has to happen right after dna replication after S phase

A

The replicated dna has to be quickly packaged into nucelosome

117
Q

How do you find if

all old histones are lost and only the new ones are assembled into nucleosomes?

A

Can get differentially label the histones with radio isotopes

Found that as soon as replication fork forms for replication, the histone start to disassemble from the template strand

The H2A and H2B dimer are released from the original DNA (free floating) and competes with the H3 H4 for binding

But the H3 H4 tetramers stay attached to the dna and randomly segregate to one of the strands (not free floating)

118
Q

In the end what was the answer to if histone are newly made or reused during replication

A

Mix of both old and new H3 and H4 / H2A H2B

119
Q

How are nucleosome modification inherited during replication

Why is this important

A

The one old histone has a modification, it recruits the enzymes that modify the new histones in the same way

Maintains the epigenetic marks from one generation to the next

120
Q

What are histone chaperones

What is their charge

A

Negative charged proteins that complex with histones and allow them to assemble into nucelosomes during replication

121
Q

What are histone chaperones

A

Negative charged proteins that complex with histones and allow them to assemble into nucelosomes during replication

122
Q

How do chaperones actually allow replication

A
123
Q

How do chaperones actually assemble nucelosomes

A

After replication of a region of dna, The histone chaperones are recruited to the ring shaped DNA sliding clamp PCNA (on the DNA)

they dissolve the PCNA right after replication is finished

Then they being and transfer the histone subunits to the newly replicated DNA

124
Q

How can geneticist see which nucleotide in DNA are modified

A

Whole genome bisulfite sequencing (WGBS)

The bisulfite only works on unmethylated cytosines

So if the DNA is unmethylated, the bisulfite comes in a removes the amino group (deaminates) the cytosine

This turns the unmethylated cytosine into Uracil.

Compare this new data to the original dna and see that the unchanged cytosines were methylated

125
Q

How can we see the interactions between genes

A

HI-C expeiments

126
Q

What is HI-C sequencing

A

Get a 3D view of the chromosome interactions in the genome

They cross link the dna using a cross linker (so the dna stays intact)

They lyse the cells, then digest the DNA into small fragments by using Use restriction enzymes to make a sticky end

Need to refill the sticky ends into one end with biotin then close the dna by ligation

Through sonicator they make it into even smaller fragments

They pull down the fragments by the biotin binding to striptoavodine

Then they sequence these fragments

Sequence Tells us the two closely neighboured chromatins that interact with each o the and can see the whole genome architecture of the chromatin structure

Cmmb 411 (6 decks)

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