Eukaryotic genome organisation

Question 1

DNA Packaging

Answer 1

DNA in a human cells ~2m in length; needs to be stored in a cell which is 10uM in length
* DNA stored with proteins
* DNA + protein = chromatin
* Chromatin =
* 1/3 DNA
* 1/3 histone proteins
* 1/3 non-histone proteins
* Chromatin is packaged into chromosomes
* Each chromosome is a single, long molecule of DNA

Question 2

Histone Proteins

Answer 2

• Small basic, positively charged amino acids (lysine & arginine)
• Make up ~half of all chromatin protein by weight
• Histones bind & neutralize negatively charged DNA

Question 3

Five types of histones: H1, H2A, H2B, H3, and H4

Answer 3

core histones (H2A, H2B, H3 + H4) form a nucleosome
* Histones are joined by linker histone, called H1

Question 4

Post translational modifications

Answer 4

Histones undergo post-translational modifications (e.g. acetylation, methylation, phosphorylation, ubiquitination) which influence chromatin function

Question 5

Non-Histone Proteins

Answer 5

1. Large variety (200 – 2,000,000) of non-histone proteins
2. Have many functions:
   * Scaffold; backbone of chromosome
   * DNA replication: e.g. DNA polymerase
   * Chromosome segregation: e.g. motor proteins of kinetichores
   * Transcriptional regulation: largest group & regulates transcription
   during gene expression; 5000 – 10 000 different transcription factors
3. Occur in different amounts in different tissue

Question 6

Nucleosomes

Answer 6

• The fundamental unit of chromosomal packaging
• Condensed DNA form chromatin fibres (string) with beads (nucleosomes)
• chromatin fibres have diameter of 20 Å
• Nucleosomes have diameter of ~ 100 Å

Question 7

More nucleosomes

Answer 7

Each nucleosome is comprised of ~ 160 bp of DNA wrapped twice around a core of 8 histones
* Linker DNA links together nucleosomes and is ~ 40 bp of DNA
* Nucleosomes are then supercoiled for more compaction (300 Å)

Question 8

Radial Loop-Scaffold Model

Answer 8

DNA compaction is sequential and progressive:
Chromatin fibres (20Å) are wrapped around nucleosomes (100Å), which are supercoiled (300 Å)
The supercoil chromatin undergoes progression compaction and forms loops held together by non-histone and scaffold and non-scaffold proteins
Chromatin loops form a rosette shape held together by additional non-histone scaffold proteins – a higher level of chromosomal compaction
Rosettes condense into compact bundles to rodlike chromosomes – this is 10 000 times more compact than naked DNA

Question 9

Chromosome Organisation

Answer 9

Each chromosome contains a single, long molecule of DNA
DNA content for each chromosome varies
Larger chromosomes have more DNA
Each chromosome has millions of base pairs of DNA
Note 1 Mb of DNA = 1 000 000 bp of DNA

Question 10

Karyotyping

Answer 10

An individual’s complete set of chromosomes

Question 11

G banding

Answer 11

Chromosomes stained with Giemsa have alternating bands of light and dark staining known as G banding
Each band contains many DNA loops and ranges from 1 to 10 Mb in length

Question 12

karyotype

Answer 12

Fully compacted chromosomes that have an unique, reproducible banding patterns
Taken during metaphase when chromosomes and sister chromatids can be seen
Karyotypes are highly reproducible within a species & within an individual throughout it’s lifetime

Question 13

1. Locate genes on a chromosome

Answer 13

A chromosome is divided into p arm (short arm) and q arm (long arm)
Both arms are divided into light/dark that are numbered consecutively
e.g CFTR gene: chromosome 7; q arm; region 3; band 1, sub-band 2 (7q31.2)
x

Question 14

Why are karyotypes useful?

Answer 14

1. Locate genes on a chromosome
2. Reveal cause of genetic diseases e.g. Edward’s Syndrome (3 copies of Chromosome 18)
3. Analyze chromosomal differences between species

Question 15

Specialised Chromosomal Elements

Answer 15

There are specialized chromosomal elements that ensure accurate replication and segregation of chromosomes:
Origins of replication
Telomeres
Centromeres

Question 16

Origins of Replication

Answer 16

Origins of replication are accessible regions of DNA with NO nucleosomes
At origins of replication DNA polymerases and other proteins initiate synthesis of DNA
Replication unit (replicon) – DNA running both ways from one origin to the endpoints and replicons are AT rich
There are many origins of replication are active at the same time
Replicons are scattered throughout the chromatin 30–300 kb apart
Rate of DNA synthesis in human cells ~ 50 nt/sec, so mammalian cells have ~ 10,000 origins, otherwise it would take 800 hrs to replicate the human genome if there 1 origin of replication!

Question 17

Telomeres

Answer 17

Telomeres are protective caps on at the end of chromosomes
They consist of DNA + proteins

Question 18

Function of Telomeres

Answer 18

preserve the integrity of linear chromosomes
prevent fusion with other chromosomes
There are NO GENES present in telomeres
They consist of TTAGGG repeats repeated 250 – 1500 times; repeat number varies in cell types
This region is bound by specific proteins that protect the ends of linear chromosomes from attack by exonucleases (enzymes that break down nucleic acids)
Telomerase enzyme prevents telomere shortening be adding on TTAGGG repeats

Question 19

Centromeres

Answer 19

Centromeres appear as constricted regions on chromosomes
They consist of blocks of repetitive, noncoding sequences = satellite DNA
Satellite DNA consists of short sequences 5-300 bases in length

Question 20

Function of Centomeres

Answer 20

Hold sister chromatids together and includes the kinetochore
Kinetochore
Structure composed of DNA + protein that helps power chromosome movement
associated with the spindle fibres during mitosis/meiosis

Question 21

Heterochromatin

Answer 21

Darkly stained regions of chromosome and highly compacted (even during interphase)
Usually found in regions near centromere
Constitutive heterochromatin: condensed most of time in all cells (e.g., Y chromosome)
Facultative heterochromatin: condensed in only some cells & relaxed in other cells (e.g. position effect variegation, X chromosome)

Question 22

Euchromatin

Answer 22

Lightly stained regions of chromosomes
Contains most genes and is actively transcribed

Question 23

Chromosomal Packaging

Answer 23

Chromosomal packaging influences gene activity

Chromosomes must first de-compact before gene expression
De-compaction is limited by boundary elements

Nucleosomes unwind to allow initiation of transcription
Transcription factors unwind nucleosomes & dislodge histones at 5’ end of genes
Unwound portion is open to interaction with RNA polymerase which can recognize promoter and initiate gene expression (transcription)

Question 24

BARR Bodies

Answer 24

Heterochromatin can silence gene expression
BARR bodies in females are an example of heterochromatin that decreases gene activity
One copy of X chromosomes is randomly inactivated in each cell
The inactivated X chromosome appears as a darkly stained heterochromatin mass in interphase cells; an example of facultative heterochromatin

Question 25

Chromosomal Rearrangements

Answer 25

Chromosomal rearrangements & changes in chromosomal number have reshaped eukaryotic genomes over evolutionary time Evolutionary paradox: There is close gene similarity over 1000’s bp between mouse & human (called syntenic blocks), but the two species have very different karyotype We can “re-construct” the mouse genome by breaking human genome into ~340 fragments (~16Mb) & pasting in a different order

Question 26

Chromosomal Deletions

Answer 26

Deletions = Removal of a segment of DNA Small deletions affect fewer genes vs. large deletions that affect 10 to 1000’s genes Phenotypic consequences of deletions: Deletion Homozygotes are often lethal due to total loss of genes Deletion Heterozygotes are often detrimental because there is a decrease in “dose” of gene & a visible phenotype Humans can’t survive as deletion heterozygotes with >3% of genome is deleted!