DLA 2-Chromosomal Organization Flashcards
Describe the enclosure of eukaryotic DNA
Eukaryotic DNA is isolated from the rest of the cell within a nuclear envelope called the nucleus
How much of the eukaryotic cell volume is taken up by the nucleus/eukaryotic DNA?
About 10% of cell volume
How much of the nucleus does the nucleolus occupy?
25% of the nucleolus
What is the purpose of the nucleolus?
Has central role making in making ribosomal RNA and ribosomal proteins
Detail the structure of the nuclear envelope
- is formed by lipid membranes
- contains large nuclear pores to transport molecules in and out
- Outer membrane also forms the endoplasmic reticulum
- nuclear lamina is fibrillation network that is associated with the inner membrane and provides stability
- heterochromatin is found near the lamina
What is heterochromatin?
Condensed DNA which is inactive, not being transcribed and is accessible to RNA polymerase
Chromosomes in interface are often referred to as _____________
Chromatin
What is euchromatin?
This is the active DNA , actively being transcribed into RNA and is accessible to RNA polymerase
Which is more abundant, heterochromatin or euchromatin ?
Most chromosomes are inactive in interphase, so heterochromatin is the most abundant
Where are chromosomes in interphase?
In interphase, they occupy chromosome territories.
List the general steps in how interphase DNA organize into the discrete chromosome structures for mitosis
- Formation of nucleosomes
- Formation of 30nm fiber
- Formation of 300nm fiber
- Condensed Chromosome 700nm
- Mitotic Chromosomes
What are histones?
Proteins which interact with chromatin
Name each histone protein
Various histone isoforms form an octamer
2 of each that form the octamer: (H2A, B , H3 and H4)
How are nucleosomes formed?
- DNA wraps twice around the octamer and thus is called a nucleosomes
- Nucleosomes have spacer DNA plus the H1 isoforms histone
- Can be refferred to as “beads on a string”
What is the Lysine-Arginine content and molecular weight of H1?
H1 is Lysine rich with a molecular weight of 23,000 Da(highest out of all histone isoforms)
What is the Lysine-Arginine content and Molecular weight of H2A histone protein ?
Slightly lysine-rich and molecular weight of 14,000 Da
What is the Lysine-Arginine content of the H2A protein and it’s molecular weight?
H2B is slightly Lysine rich and has a molecular weight of 13,800Da
What is the Lysine -Arginine content and molecular weight of H3?
H3 is Arginine rich with MW of 15,300
What is the Lysine-Arginine content and molecular weight of H4 histone isoforms ?
H4 is Arginine rich and molecular weight of 11,300
What is usually the positive charge of H3 and why?
H3 usually has a charge of +14, close interaction with the negatively charged DNA
Who performed x ray crystallography on the histone DNA nucleosome?
Richman and colleagues
How do histone tails affect the DNA helix?
- Histone tails protrude into the minor groves of the DNA helix
- Amino acids on the histone tails can be chemically modified to change how the tails interact with the DNA
Positively charged amino acids like Lysine can be acetylated, what effect does this have ?
Acetylation will interrupt ionic bonding between lysine and DNA, resulting in a more relaxed interaction
What are the most commonly phosphorylated amino acids?
Serine, threonine and tyrosine
What amino acids can be phosphorylated?
Serine, threonine and Tyrosine -most common
But also arginine, lysine, aspartic acid, glutamic acid and cysteine
What effect that some amino acids can be phosphorylated on the ability of positively charged histone tails to interact with negative charged DNA?
Phosphorylation of histone tails reduces the positive charges on the histone tail, can occur during particular times of the cell cycle such as mitosis which decreases the positive charge on histone tails
Describe the formation of 30 nm fiber
- beads on string formation will further condense to a 30 nm fiber
- H1 histones(purple) associate to form the core of the 30nm fiber, also referred to as the solenoid structure
What role did Robinson play in understanding the 30nm fiber formation?
Robinson and coworkers reconstituted 30 no fibers in vitro and performed X-ray crystallography to characterize the packaging of DNA
Describe the formation of 300 Na fiber
- the 30 nm fibers are folded into large loops of interphase chromatin
- Can be refferred to as “bottle brush” or “rosettes of chromatin loops”
- Non-histone proteins form a scaffold for the chromatin in the nuclear matrix
- the diameter of the chromatin fiber is now 300 nm
- this DNA can still be replicated or transcribed to RNA
How much base pairs are contained in each bottle brush/rosettes of chromosome loops/ loops in 300 no fibers?
Each loop contains between 20,000 to 80,000 base pairs
Can DNA in a 300 no fiver be replicated or transcribed to RNA?
Yes
Describe the formation of condensed chromosomes- 700 nm
Beginning in early mitotic cell cycle, the chromatin is in its most compact state
- gene expression shut down
- histones are reorganized to help reorganize chromatin
- Condensins are protein complexes (Smc4 and Smc2) which help interphase DNA produce sister chromatids of a mitotic chromosome
- ATP is used as energy
- about 1 condensin per 10,000 bp
- SMC- Structural maintenance of chromosomes
When is gene expression shut down?
In formation of 700nm fibers
When are histones are reorganized into chromatin?
In the formation of 700nm condensed chromosomes fibers
Describe the formation of mitotic chromosomes
- Sister chromatid are held together by Cohesin protein complexes
- Cohesin: Smc1, Smc3, Scc3 and Scc1(sister chromatin cohesion complex)
- Cohesin form giant rings around the two sister chromatids during S phase and remains till the transition from metaphase to anaphase
Describe heterochromatin in detail
- interphase chromatin remains densely packed
- transcriptionally inactive
- DNA in the centromeres region and the telomeres region typically remains densely packed
- inactive satellite sequences are heterochromatic
- AT rich and is darkly stained by Geimsa
How does Geimsa stain react with heterochromatin?
Produces a dark stain
Describe euchromatin in detail
- during interphase, most chromatin is decondensed or loosely packed
- Potential to be transcriptionally active
- GC rich and does not take up Geimsa stain well
How does Geimsa stain react with euchromatin?
Doesn’t take up Geimsa well
Where are specialized eukaryotic chromosomes found?
Found in specific eukaryotic cells
How big are specialized eukaryotic chromosomes?
Very large such as that they could be seen using a light microscope in the 1880s
Which studies of specialized chromosomes have insight to genetic information and structure/arrangement?
- Polytene Chromosomes and the Puff regions 1881 E.G. Balbiani
- Lamp brush Chromosomes 1882 Walther Flemming
Where are polytene chromosomes found?
In insect larvae, protozoans and helminths
When can Polytene Chromosomes be found?
During interphase
How large are polytene chromosomes?
Polytene chromosomes is 200-600 um long and can be seen with light microscope
What do polytene chromosomes appear as?
- large regions of paired homologs (rare in somatic cells) which undergo many rounds of replication without separating or cell division
- linear series of alternating bands (B) and interbands (IB) “chromomeres’
- DNA becomes uncoiled and makes a bulge that looks like a “Puff” which has high levels of RNA transcription
Describe the appearance of lampbrush chromosomes
- Homologous pairs of chromosomes held together at Chiasmas
- Extensive looping of DNA from 500-800 um
- Later in meiosis the loops return to normal lengths of 15-20 um
Describe the activity of Lampbrush chromosomes
They are transcriptionally active chromosomes in large oocytes in first prophase of meiosis (vertebrates and invertebrates but not mammals)
What are the 3 characteristics of Repetitive DNA sequences?
- Do not code for proteins
- Many are transcribed to RNA
- May be involved on chromatin remodeling
What are the 3 broad classes of repetitive DNA?
Satellite DNA
Tandem repeats
Interspersed retrotransposons/ transposable sequences
What is satellite DNA?
Heterochromatin repetitive sequences we find flanking the centromere- this means in the centromeres region either a little to the left or right
What are tandem repeats?
Can either be short or long tandem repeats, lined up in a tip to tail fashion
What are interspersed retrotransposons/transposable sequences?
Found throughout the chromosomal DNA/genome and have the ability to transpose themselves.
This means, they can translated to RNA and then reversed back to DNA and transposed from one region to the other of a chromosome
About how much of the human genome consists of Repetitive DNA?
About 50% of human genome consists of repetitive DNA sequences with or without transposable elements
How was satellite DNA initially studied?
Sedimentation equilibrium Centrifugation (in CsCl) to determine destiny, plotted concentration of DNA vs. destiny
Describe the density of DNA
Most DNA is uniformly dense (MB) with a narrow range of GC:AT of about 41%
DNA with a different GC:AT ratio= different density(S)
What was found of satellite DNA after further analysis?
After further analysis than sedimentation equilibrium Centrifugation, it was found satellite DNA is :
- composed of short, repetitive sequences found in heterochromatic DNA
- contributing to chromosomal organization
- helps at Centromeric regions of chromosomes(association with unique histone 3 isoforms at kinetochore region)
- helps at telomeric regions of chromosomes(repetitive regions to protect the ends of chromosomes from degradation)
Name 2 types of Moderately repetitive DNA
- Variable number tandem repeats(VNTRs), dispersed throughout genome
- Short tandem repeats(STRs), dispersed throughout genome
Describe Variable number tandem repeats (VNTRs)
- 15-100 base pairs long
- Tandemly repeated 5-100 times
- Repeat size varies for individuals
- “Minisarellites”
Describe Short tandem repeats (STRs)
- di, tri, tetra and pentanucleotide
- Tandemly repeated 5-50
- repeat size varies for individuals
- “Microsatellites”
What is the bases of fingerprinting?
Examination of many VNTRs and STRs from an induvidual
What are SINES and LINES?
-transposable sequences that are mobile, can relocate within genome
How are SINES and LINES able to be transposed?
Sequence contains information to make enzyme Reverse Transcriptase
- Reverse transcriptase can convert its own RNA into DNA which can then integrate somewhere else in the genome
How do SINES and LINES differ from tandem repeats?
SINES and LINES are dispersed throughout genome and not Tandemly repeated
How much of the human genome constitutes SINES and LINES?
Constitutes 1/3of the human genome
Describe SINES
- short interspersed elements
- <500 bp up to 1,500,000Xs
What are LINES ?
- long interspersed elements
- ~6000 bp to 100,000 Xs
What are pseudogenes?
Large number of single copy non coding regions
What do Pseudogenes represent?
Represent evolutionary vestiges of duplicated copies of genes that have undergone significant mutational alteration, insertions and deletions
Why do pseudogenes not code for anything?
-Some homology to the parent gene but are not transcribed, thus does not make a protein product
What is the most likely cause of psuedogenes?
Most likely due to reverse transcriptase copying RNA to DNA reinsertion into the genome
Describe Viral and bacterial genomes
- Single nucleic acid molecule(linear or circular).
- RNA or DNA
- Single stranded or double stranded
What is the method for packing of viral genome?
Mechanism for packing of viral genome continues to be investigated
What is the method file packaging of bacterial genome ?
Mechanism for packing of bacterial genome assisted by proteins
What does viral life cycles depend on?
The life cycle of viruses depend on host cell for propagation
The viral genome completely fills the…
Caspid
What does the virus need for its life cycle specifically?
The virus needs genomic information packed within a protein
What happens once a virus invaded a host cell?
Once a virus invaded a host cell, it hijacks the host cell to duplicate viral genome, synthesize viral proteins and assemble many new viral particles
-Mechanism for compaction of the genome still unclear
Give the details of SS RNA compaction in viruses
- still unclear, but recent fluorescent experiments suggests the following mechanism:
- ssRNA forms secondary helical structures
- Polycations, like spermidine, found within the host cell may surround the RNA and invoke condensation
- Some of the RNA helixes have binding affinity to viral coat proteins (CP)
- When the host cell makes an abundance of coat proteins, they begin to displace the polycations
- The coat proteins assemble into the complete virus, completely full of genomic material
Describe the appearance of double stranded viral RNA
- similar packaging for phage and viruses with double stranded genome
- Appears like a ball yarn wrapped around the center
- Phage dsDNA terminus is threaded through the core to be injected into host bacterial cell
Where does the circular double strand DNA compact?
Circular double strand bacterial DNA becomes compacted within the nucleoid
Name two middle repetitive DNAsegments
Tandem repeats
Interspersed retrotransposons(SINES and LINES)
Name repetitive DNA that would be considered highly repetitive DNA
Satellite DNA
Name the types of repetitive tandem repeats DNA
- Mini satellites-VNTRs
- Microsatellites- STRs
- Multicopy genes- rRNA genes
Give the types of interspersed retransposons
SINES-Alu
LINES-L1
How does bacterial DNA form a nucleoid?
HU proteins(heat unstable) and H-NS(histone-like nucleoid structuring ) associate with the DNA and form a multi-protein assembly within the bacteria, forming the nucleoid
HU an H-NS proteins are basic (positively charged at pH 7)
What is the purpose of Supercoiling?
Supercoiling of the DNA stabilizes base-pairing when under-winding occurs in a region during transcription or replication
Describe the discovery of DNA Supercoiling
In the early 1960s, ultracentrifugation experiments of circular bacterial DNA found three distinct forms of circular DNA, though all had the same molecular weight but differred in density and compactness
When does DNA underwinding occur and what effect does underwinding have ?
DNA underwinding occurs during transcription and replication and interferes with normal base-pairing
How much of the eukaryotic genome encodes for proteins?
About 2% encode for proteins
What is viscosity and it’s relation to DNA absorbsion?
Viscosity-fluidity thickness
Inversely proportional to DNA absorption in spectrometer
Because single stranded DNA absorbs more
