Chapter 10 Flashcards
What happens in the 1st level of organization of DNA packaging in to chromatin?
forms 10nm fiber involving nucleosomes
What happens in the 2nd level of organization of DNA packaging in to chromatin?
superhelical coiling of nucleosomes to generate a 30nm fiber
What happens in the 3rd level of organization of DNA packaging in to chromatin?
looping the 30nm fiber to generate a 300nm fiber involving interphase matrix proteins
What happens in the 4th level of organization of DNA packaging in to chromatin?
chromosome condensed and form 700nm per sister chromatid
What happens in the 5th level of organization of DNA packaging in to chromatin?
forms 1400nm for entire metaphase chromosome
Where is chromatin?
spread out in nucleus
What chromatin occupies most of the nucleus?
euchromatin
Which chromatin is more densely packed?
heterochromatin
Heterochromatin is similar to what?
chromosome at mitosis densely packed
Where is heterochromatin found at?
centromeres
What does heterochromatin form?
forms discrete clumps and aggregate into a chromocenter
Constitutive heterochromatin?
region that are always heterochromatic
What does constitutive heterochromatin contain?
multiple copies of non-transcribed repeats
How many and what kind of genes do constitutive heterochromatin have?
few genes and inactivated genes
Facultative heterochromatin
euchromatin regions converted to a heterochromatic state
What are 3 events that are associated with causing the condensation of euchromatin to heterochromatin?
a. reduced acetylation of histone proteins
b. increased methylation of histone proteins
c. hypermethylation of cytosine bases in DNA
What is Giemsa stain used for?
for staining process to generate G-bands allowing each chromosome to be identified by its characteristic banding pattern
What has lower GC content and what has higher GC content?
bands have lower GC content and interbands have higher GC content
Genes tend to be concentrated in regions of?
higher GC content (interbands)
Where is the 4th level of chromatin organization/packaging found?
in some heterochromatin and in mitotic chromosomes
DNA fiber is arranged into a series of?
loops/domains
The loops/domain of DNA fiber is attached to what?
to a central proteinaceous ‘matrix’
What is used to intercalate between base pairs and generate positive superhelical turns in ‘closed’ circular DNA molecules?
Ethidium bromide
Ethidium bromide has allowed researchers to estimate what?
the size of the DNA looped domains
How are the ends of the looped domains in the E. coli chromosome distributed?
randomly distributed
What are the two DNA sites attached to proteinaceous structures in interphase nuclei?
MARS = matrix attachment region
SARS = scaffold attachment regions
MARS
matrix attachment regions
SARS
scaffold attachment regions
The DNA sites attached to the proteinaceous matrix is necessary for?
transcription or replication
The interphase matrix is related to what?
mitotic scaffold
MAR fragments are attached to what two?
interphase matrix and mitotic scaffold
What is a prominent component of the chromosome scaffold and is a part of the nuclear matrix?
Topoisomerase II
MTOC
microtubule organizing center
Where does MTOC exist?
at the poles of the cell and on each chromosome
MTOC on each chromosome is part of what?
kinetochore
What is a good evidence that the centromere is essential for segregation of chromosomes into progeny cells?
loss of acentric fragments during miosis/meiosis
What are visualized as constrictions?
centromeric DNA
Microtubule binds to what?
MBPs = microtubule-binding proteins
MBPs bind to what?
CBPs = centromeric-binding proteins
MBPs
microtubule-binding proteins
CBPs
centromeric-binding proteins
CBPs bind to what?
centromere DNA
When does cohesin begin to degrade?
anaphase
Any stretch of DNA capable of centromeric (CEN) function should confer what?
mitotic stability upon a plasmid - help it segregate properly to progeny cells
Yeast CEN consensus is ____-rich
A-T rich
Knowledge of functional CEN sequences is essential for?
construction of YACs and HACs
Short sequences of DNA repeated many times in tandem (no coding function) are often found in?
putative centromere regions
tandem
no coding function
For primates, tandem arrays of a 170 ntp repeating unit is known as ?
alpha satellite DNA
How long is the tandemly repeating units in vertebrates?
6 nucleotides long
What is the human telomere repeating unit sequence?
3’ CCCTAA 5’
Telomeres have what tail?
ssDNA G-tail
How long is the ssDNA G-tail?
14-16 nucleotides long
Telomere tails are ____-rich strand
GT
What is a telomerase?
ribonucleoprotein enzyme
Telomerase carries ___________ with the same sequence as _________ strand
RNA template; CA-rich
Function of telomerase (2)
a. adds repeats to a telomere by binding its RNA to the GT-rich strand
b. extends the GT-rich strand with reverse transcriptase activity
End of chromosomes are stabilized by forming what?
long loop of DNA
In animal cells, how long is the loop of DNA at the end of chromosomes?
5-10 kb
What displaces a same sequence region upstream in the telomere?
3’ ssDNA end (GT-rich)
The ss region pairs with what?
with its homologous strand
What special chromosome have allowed researchers to visualize gene expression in its natural state?
lampbrush chromosome of amphibians
When are lampbrush chromosome of amphibians formed?
during an extended meiosis
Structure of the lampbrush chromosome
stretched out
many lateral loops that extrude from chromomeres
loops extended in pairs, one from each sister chromatid
What does the loops of the lampbrush chromosome contain?
nascent RNA chains
Increase in the ____ length of the lampbrush chromosome loops indicates ______.
RNP; transcription unit
Other than the lampbrush chromosome, what is an another special chromosome?
polytene chromosome
Where are polytene chromosomes found?
in dipteran insect larvae; in interphase nuclei of salivary glands and imaginal discs
The polytene chromosome was created due to?
multiple rounds of replication without mitosis
Polytene chromosomes consist of _______ when their centromeres aggregate to form a ___________?
4 synapsed diploid pairs; chromocenter
What contains most of the DNA mass?
Bands
________ stain more lightly
interbands
Most or all bands contain more than _____ gene
1
What can illustrate the position of specific genes on the cytological map?
In situ hybridization with labeled probes
A transcriptionally active region
chromosome puff
What happens at the chromosome puff?
chromosome fibers unwind from their usual state of packing in the band
Very large puff that represents more than one gene being actively transcribed
Balbiani ring
Pattern of puffs is related to what?
gene expression
Puffing pattern changes during
larval development
Nucleosome is composed of
8 histone proteins (2 each of H2A, H2B, H3, and H4) plus ~2 wraps of DNA
Treating chromatin with micrococcal nuclease causes
release of individual nucleosomes and destroys the lnker DNA between them
A ladder of DNA fragments
Micrococcal nuclease
Each step of the ladder represents
DNA derived from a discrete number of nucleosomes
How much of the DNA chromatin is in the form of the 200 bp ladder?
> 95%
What has 146bp of DNA?
nucleosome core particle
Nucleosome core particle has 146bp DNA associated with what?
histone octamer
Micrococcal nuclease first cuts between what?
adjacent nucleosomes
Mononucleosomes are associated with how many bp of DNA?
200
If micrococcal nuclease is allowed to continue digesting, _______ will have what length of DNA?
trimmed nucleosome; DNA length of 165 bp
If micrococcal nuclease is allowed to digest more then the final protected DNA length is?
146 bp
DNA length of each nucleosomes:
mononucleosomes -
trimmed nucleosomes -
core particles -
mononucleosomes - 200 bp
trimmed nucleosomes - 165 bp
core particles - 146 bp
What is relatively resistant to nuclease digestion
nucleosome core DNA
What comprises the rest of the variable DNA length of the repeating unit?
Linker DNA
Structure of the major part of the core DNA on the nucleosome
tightly curved on the nucleosome
Structure of the terminal and linker regions on the nucleosome
extended on the nucleosome
What is located in the region of the linker DNA immediately adjacent to the core DNA?
Histone H1
Which enzymes make single-strand nicks in DNA?
DNAase I and DNAase II
Which histone proteins form a tetramer?
H3-H4
Tetramer binds to what two dimers? forming what?
H2A-H2B dimers to form the octamer
The binding of DNA to histone proteins is via what?
sugar-phosphate backbone
What of histone proteins extend away from the nucleosome core?
N-terminal tails
N-terminal tails of histone proteins emerge between what?
DNA turns so they are available for modification
10-nm fiber of chromatin structure
string of nucleosome beads
10-nm fiber of chromatin does not require what for its structure
histone H1
30-nm fiber of chromatin structure
coiling of 10-nm fiber into a solenoid with the assistance of histone H1
Replicated DNA in a replication fork is immediately incorporated into?
nucelosomes
Progress of replication fork disrupts the nucleosomes, then they from on what?
daughter duplexes
What associates with the replication fork?
CAF-1
What binds to H3 and H4?
CAF-1
What helps H3 and H4 from a tetramer?
CAF-1
What helps add the H2A and H2B dimers?
CAF-1
What experiment involve heavy vs. light amino acid switching?
Meselson-Stahl type experiment
Experiments with heavy vs. light amino acid switching illustrates what?
that nucleosomes are a mixture of old histone proteins and new histone proteins, so some dissociation and reassociation of the components of the octamer must occur as replication fork moves through
Type of organization where each site on the DNA always is located a particular position on the nucleosome
Nucleosome positioning
What technique was used to determine if nucleosome positioning is used or if placement of nucleosomes is random along the DNA
indirect end labeling
DNA is cleaved with what two?
m-nuclease and restriction endonuclease
If nucleosome positioning is used, the probe would only bind to what on the gel?
single fragment
The restriction endonuclease cutting site will be in a variable position relative to where m-nuclease cut if the position is ____.
random
A smear of bands of variable sizes would bind to ?
probe
What two mechanisms does nucleosome positioning occur?
intrinsic and extrinsic
Describe the intrinsic mechanism.
nucleosomes are deposited specifically at a particular DNA sequence
structural DNA features help the positioning
Describe extrinsic mechanism
region excludes nucleosomes, serves as a boundary from which a series of nucleosomes are assembled sequentially.
Results from the interactions of other proteins with the DNA and histones
What are regions of chromatin which first experience breaks when bery low [DNAase] are used?
hypersensitive sites
Hypersensitive sites are not arranged in the typical nucleosomal structure, therefore, _______.
DNA is not protected
Hypersensitive regions are what regions?
regulatory regions for gene expression
hypersensitive regions lack
histone octamers
Genes which are able to be transcribed in a given tissue are preferentially susceptible to what?
DNAase I especially their regulatory regions - promotors, enhancers
Mostly transcribed genes probably retain their _____.
nucleosomes
How are most transcribed genes structures change as RNA polymerase passes?
temporarily displaced as RNA polymerase passes through but re-form immediately afterwards
For what genes is the transcribed region is devoid of nucleosomes?
intensively transcribed genes (ex. for rRNA)
When is DNA displaced from around the core histones?
As RNA polymerase enters the nucleosome
How are nucleosomes assembled from one position to another?
RNA pol binds at the promoter, advances, displaces octamer, and transcribes up to the terminator. Octamer reinserted behind pol.
What is repositioned on DNA behind RNA pol at a new site?
histone octamer
What does transcription tends to destroy?
nucleosomal positioning
If transcription is repressed, what happens to the nucleosomal positioning?
reestablished within 10 mintues
Adding _____ to the experimental system results in _____.
histone H1; slowed down transcription
What is necessary for the removal and assembly of octamers?
remodeling complexes
Remodeling complexes are recruited by _____ and require _______ to change chromatin structure.
transcription factors; ATP energy
FACT
Facilitates Chromatin Transcription
Structure of FACT
heterodimer
Function of FACT
a. Associates with RNA pol II to cause nucleosomes to lose H2A and H2B dimers
b. helps add H2A and H2B dimers to a nucleosome that is forming following transcription
c. used during DNA replication and repair
What is considered to be a transcription Elongation Factor?
FACT
What is associated with the chromatin of active genes?
FACT
What refers to the repression of gene activity in a local chromosomal region?
silencing
Changes in where may be either activating or repressing?
at an individual promoter
Acetylation of histones is often associated with what?
gene activation
Deacetylation of histones is one way to do what?
repress transcription
What induces changes in chromatin structure and requires energy in the form of ATP hydrolysis?
Chromatin remodeling
What is the most common use of chromatin modeling?
change the organization of nucleosomes at the promoter of a gene that is to be transcription
When is chromatin remodeling needed?
replication and damaged DNA repair
When does the histone octamer moves along DNA without ever losing contact with it?
sliding reaction of some remodeling mechanisms
CRCs
Chromatin Remodeling Complexes
How are chromatin remodeling complexes targeted to specific chromatin sites?
by being recruited by activators or repressors
Chromatin structure is affected by what kind of covalent histone modifications?
modifying the N-terminal tails of H3 and H4
What kind of modifications create binding sites for the attachment of nonhistone proteins that change the properties of chromatin?
methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation
Modifications create binding sites for what?
for the attachment of nonhistone proteins that can change the properties of chromatin
What modification is associated with active chromatin?
Acetylation
What modification is associated with inactive chromatin?
Methylation
What is required prior to the assembly of nucleosomes during replication?
transient acetylation of histone H3 and H4
What catalyzes the acetylation of histones?
histone acetyltransferase
HATs
Histone acetyltransferases
Group A HATs act on which histones and what is their function?
acts on histones in chromatin; help control transcription/gene expression
Group HATs act of which histones and what is their function?
acts on newly synthesized histones in the cytosol during S phase
What catalyzes the removal of acetyls from histones?
Histone deacetylases
HDACs
Histone deacetylases
Widescale changes in acetylation occur on?
sex chromosomes
What chromosomes has underacetylated H4?
female mammal inactive X chromosome
What chromosome has increased acetylation of H4?
super-active X chromosome in Drosophilia
Recruitment of _____ or _____ to specific targets is likely to be indrect through activators or repressors
HATs; HDAC
Inactivation spreads from ______ to _____.
heterochromatin into the adjacent region
What results from inactivation spreading form heterochromatin to adjacent region?
position effect variegation
Inactivation is considered an ____ and happens in ______.
epigenetic effect; in embryonic development
What is telomeric silencing?
genes translocated into a telomeric location show loss of activity due to the spreading effect that the propagates from the telomeres
Interaction of heterochromatic proteins is due to ?
interactions with the N-terminal histone tails
HPs
heterochromatic proteins
When an H3 Lys is deacetylated what binds and what results from that?
HP1 binds and a different H3 Lys is methylated
Some HP proteins interact with histones after they interact with what?
DNA-sequence-specific binding protein
RAP1 binds to what?
to DNA repeats at telomeres and to SIR3 & SIR4
SIR3 and SIR4 interact with what?
N-terminal tails of H3 and H4
Unequal number of sex chromosomes necessitates the need for what?
dosage compensation
Dosage compensation serves to
equalize the level of expresion of X-linked genes into the two sexes
What includes sequences with no or very little coding function?
constitutive heterochromatin
What includes coding sequences?
Facultative heterochromatin
Xic
X-inactivation center
What is Xic?
x-chromosome locus responsible for inactivation
Function of Xic
counts X-chromosomes and inactivates all copies but one
Xist
X-inactivation specific transcript
Where is Xist?
in Xic locus
Function of Xist RNA
coats the X-chromosomes, playing a role in inactivation
SMC
structural maintenance of chromosome
What are SMC family of proteins?
ATPases
What are the two functional groups of SMC family proteins
condensins and cohesins
What help condense chromatin into compact chromosomes at mitosis?
condensins
What connects sister chromatids, allowing them to be released at mitosis?
cohesins
