Hiroki Shibuya - Genome and chromatin structure

Question 1

Nucleosome

Answer 1

The complex between DNA and histone.

Question 2

Plasmids

Answer 2

Prokaryotes have genes on the extrachromosomal plasmids. Plasmids have genes to control its own replication, does not contain genes that are vital under all conidtions, and often carry genes that provide specific properties such as antibiotic resistence.

Question 3

What is a gene?

Answer 3

A gene is a segment of the DNA that contains information to make protein or an RNA in cases where only RNA is formed.

Question 4

Genes in eukaryotes

Answer 4

The genes are scattered one by one on the two DNA strands of the chromosomes. When transcribed, it gives rise to a messenger RNA. The number of genes in eukaryotes varies between 6000 - 25000.

Question 5

Operon

Answer 5

In prokaryotes, the genes of the same function are gathered in operons. An operon is a functional unit of DNA, containing a cluster of genes under the control of a single promoter. When transcribed, it gives rise to a polycistronic RNA; an RNA that encodes more than one protein.

Question 6

Differences between genome in prokaryotes and eukaryotes

Answer 6

Prokaryotes:
- 10^6 - 10^7 nucleotide pairs.
- Circular DNA.
- 500 - 4000 genes.
- Genes in operons.
- Polycistronic mRNA.
- Only coding sequenses in genes.
- Transcription and translation in the cytoplasm.

Eukaryotes:
- 10^7 - 10^9 nucelotide pairs.
- Linear DNA.
- 6000 - 25000 genes.
- Genes one by one.
- Monocistronic mRNA.
- Genes contain introns and exons.
- Transcription in the nucleus, translation in the cytoplasm.

Question 7

Types of repetitive DNA

Answer 7

Interspersed repeats: tRNA, pseudogenes, transposons.
Tandem repeats: rRNA and satellite DNA: microsatellites and minisatellites.

Question 8

Pseudogenes

Answer 8

Nonfunctional segments of the DNA that resembles functional genes. Nonprocessed pseudogenes and processed pseudogenes.

Question 9

Nonprocessed pseudogenes

Answer 9

Located on the same chromosome as the original gene. Possess introns. The loss of regulatory elements, premature stop codons, frameshift mutations or alterations in splice sites prevent them from being transcribed or encoding functional proteins.
Created by gene duplication.

Question 10

Processed pseudogenes

Answer 10

Created by reverse transcription.
1. A gene is transcribed into mRNA.
2. Reverse transcriptase sunthesize the complementary DNA.
3. The second DNA strand is synthesized.
4. The cDNA is inserted into the genome.
No introns. Accumulation of mutations.

Question 11

rRNA in tandem

Answer 11

Genes encoding rRNA are repeated in tandem. There are 280 rRNA genes in the genome distributed in clusters on five seperate chromosomes.
The genes for 18S, 5.8S, and 28S rRNA form a single transcription unit that is transcribed by RNA polymerase I to give a single large RNA. The longer transcript is then cleaved to release three seperate rRNA molecules.

Question 12

Tandem repeats

Answer 12

Tandem repeats occur in DNA when a pattern of one or more mucleotides is repeated and the repetitions are directly adjacent to each other.

Question 13

Minisatellite DNA

Answer 13

Minisatellite DNA (10-60 bp) are short tandem-repeated DNA sequences that are mainly found in the centromeres and subtelomeres. Important for the chromatin structure in these parts.

Question 14

Microsatellite DNA

Answer 14

Shorter than minitsatellites. Also called STR (short tandem repeats). Used when making DNA profiles (DNA fingerprints). The flanking sequence of the STR is the same, but the number of repititions differs between individuals.

Question 15

STR DNA fingerprints

Answer 15

The flanking sequence of the STR is the same, but the number of repititions differs between individuals. By looking at 10-15 positions in the genome, you get the gender and a unique set of repititions for each person.

Question 16

DNA-transposons

Answer 16

Mostly in bacteria. Contains a gene encoding transposase (enzyme that mediates cleavege of the transposon). Enables the transfer of genes between different chromosomes by cut and paste mechanism.

Question 17

Retroviral-like retrotransposons (LTR)

Answer 17

The protein coding part expresses the enzyme reverse transcriptase (RT) and integrase. The LTR includes a promoter, which means that it is expressed regardless of where it ends up. Jumps via an RNA intermediate.

Question 18

Non-retroviral retrotransposons

Answer 18

Two types: LINES (longs interspersed nuclear element) and SINES (short interspersed nuclear element). Together they make up 30% of the human genome.

Question 19

LINES

Answer 19

A non-reotroviral retrotransposon. Long repetetive sequences of about 7000 bases. Encodes an RNA-binding protein (ORF1), a reverse transcriptase and an endonuclease (ORF2). Lack promoter and must end up in a transcriptionally active area to be expressed.

Question 20

SINES

Answer 20

A non-retroviral retrotransposon. Short repetitive sequences of about 100-700 bases. Does not encode any proteins. Lacks promotes and must en up in a transcriptionally active area to be expressed.

Question 21

What is the function of a transposon?

Answer 21

In the process of insertion into the genome, transposons can:
- interrupt the coding DNA
- create gene mutations
- turn off nearby genes, preventing their ability to create proteins.

Transposons can be “selfish genes”, providing no benefit to the host.

There is a benefit for host organisms:
- increased genetic diversity
- produce novel genes

Question 22

Chromosome territories

Answer 22

During interphase, the chromatin is loosely packed. Each chromosome occupies a specific area in the nucleas, called chromosome territories.

Question 23

What does a nucleosome consist of?

Answer 23

2 x histon H2A
2 x histon H2B
2 x histon H3
2 x histon H4
147 basepairs of DNA

Question 24

Histone structure

Answer 24

Contains many basic amino acids such as lysine and arginine, which makes them positively charged. They have a long N-terminal which is modified. They are very well preserved.

Question 25

Chromatin structure dynamic, regulation

Answer 25

The chromatin can be decondensed or condensed. The ATP-dependent chromatin remodeling complexes alter the chromatin structure.
The chromatin structure is regulated by histone tail modifications. The modifications that mainly take place are: acetylation, methylation and phosphorylation.

Question 26

Effects of modifications on chromatin

Answer 26

The effect depends on which amino acids that are modified.
Acetylation increases the negative charges on the histones, weakening the DNA-histone interaction, making the chromatin less condensed.
Methylation can lead to both relaxation and condensation of the chromatin, depending on which histone is modifies.

Question 27

What creates the diversity in chromatin regulation?

Answer 27

That there are many different, specific histone variants, and that a large number of different modifications can be made to the histone tails.

Question 28

Histone code hypothesis

Answer 28

• A regulatory protein binds to the DNA.
• This recruits a histone modification enzyme.
• A reader protein binds, which recruits a new histone modifying enzyme. Positive feedback, expansion of modification.
• This is repeated until a specific chromatin structure is created.

Question 29

Barriers of chromatin modifications

Answer 29

Heterochromatin (dense chromatin) spread is prevented by:
- the chromatin sits together with another complex, like the nuclear pore complex.
- barrier proteins can interract with nucleosomes.
- a histone modifying complex has ensured that the chromatin is acetylated on the histone that would be methylated during packing in the heterochromatin.

Question 30

Inheritence of chromatin structure

Answer 30

Upon replication, the newly sunthesized strands inherit half of the nucleosomes. To the newly incorporated histones, the modifications are reesablished.

Question 31

X-chromosome inactivation

Answer 31

One of the womans X chromosomes needs to be inactivated for dosage compensation. During early embryogenesis, randomness determines which of the two X chromosomes is inactivated. Once established, the chromatin state is inherited through out life.