Section D - Prokaryotic Section D - Prokaryotic and Eukaryotic and Eukaryotic

Chromosome StructureChromosome Structure

D1 Prokaryotic chromosome structureD1 Prokaryotic chromosome structure The Escherichia. coli chromosome, DNA domains,

Supercoling of the genome, DNA-binding proteinsD2 Chromatin Structure D2 Chromatin Structure Chromatin, Histones, Nucleosomes, The role of H1, Linker DNA,

The 30 nm fiber, Highter order structureD3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure The mitotic chromosome, The centromere, Telomeres,

Interphase chromosome, Heterochromatin, Euchromatin, DNase hypersensitivityⅠ , CpG methylation, Histone variants and modification

D4 Genome complexityD4 Genome complexity Noncoding DNA, Reasociation Kinetics, Unique sequence DNA,

Tandem gene clusters, Dispersed repetitive DNA, Satellite DNA, Genetic polymorphism

D5 The flow of genetic informationD5 The flow of genetic information The central dogma, Prokaryotic gene expression,

Eukaryotic gene expression

ContentsContents

D1 Prokaryotic chromosome structure — D1 Prokaryotic chromosome structure —

The Escherichia. coli chromosomeThe Escherichia. coli chromosome

• A single closed-circular DNA, 4.6Mp

• The DNA packaged into a region known as Nucleoid that contains high concentration of DNA (up to 30-50 mg/ml) as well as all proteins associated with DNA.

• Continuous replication (no cell cycle)

D1 Prokaryotic chromosome structure — D1 Prokaryotic chromosome structure —

DNA domainsDNA domains

• Observed under electron microscopeObserved under electron microscope

• 50-100 domains or loops per 50-100 domains or loops per E. coliE. coli chromosome, with chromosome, with 50-100 kb/loop50-100 kb/loop

•The ends of loops are The ends of loops are constrained by binding to constrained by binding to a structure which a structure which probably consists of probably consists of proteins attach to part of proteins attach to part of the cell membrane.the cell membrane.

D1 Prokaryotic chromosome structure — D1 Prokaryotic chromosome structure —

Supercoling of the genome Supercoling of the genome

• E. coli chromosome as a whole is negatively E. coli chromosome as a whole is negatively supercoiled.supercoiled.

• Individual domains may be supercoiled indepIndividual domains may be supercoiled independently (topological independent). endently (topological independent).

• Direct biochemical evidence is lacking for diffDirect biochemical evidence is lacking for different level of supercoiling in different domainerent level of supercoiling in different domains. s.

D1 Prokaryotic chromosome structure — D1 Prokaryotic chromosome structure —

DNA-binding proteinsDNA-binding proteins

• Histone-like proteins essential for DNA packaging to stabilize and constrain the supercoiling.

• HU: a small basic dimeric ( 碱性双体 ) protein, non-specific binding to DNA, most abundant.

• H-NS (protein H1): neutral monomeric ( 中性单体 ), partially non-specific binding

• Site-specific DNA binding proteins important for organization of DNA domains (RNA polymerases, IHF etc).

D2 Chromatin Structure D2 Chromatin Structure — — ChromatinChromatin

• Fig. 1: The major structures in DNA compaction; DNA, the nucleosome, the 10nm "beads-on-a-string" fibre, the 30nm fibre and the metaphase chromosome.

DNA compaction nucleosome

10nm "beads-on-a-string" fibre

30nm fibre

metaphase chromosome

DNA

Chromosome(condensed)

Chromatin(diffused)

Interphase 间期

Mitosis 有丝分裂

D2 Chromatin Structure D2 Chromatin Structure — — HistonesHistones

• The major protein components of chromatin.• Four families of core histone: H2A, H2B, H3 and H4, An

additional non-core histone H1.• Small, 10 kDa for core histones and 23 kDa for H1.• Basic (rich in lysine and arginine) and tightly binds to DN

A.

Histone octamer

Top view

Side view

D2 Chromatin Structure D2 Chromatin Structure — — NucleosomesNucleosomes

• The nucleosome core is the basic unit of chromosome structure, consisting of a protein octamer containing two each of core histones.

• With 146 bp of DNA wrapped 1.8 times in a left-handed fashin around it.

• The wrapped of DNA into nucleosomes accounts for virually all of the negative supercoiling in eukaryotic DNA.

D2 Chromatin Structure D2 Chromatin Structure — — The role of H1The role of H1

• Stabilizes the point at which DNA enters and leaves the nucleosome core.

linker histone H1 and H5 family

•C- tail of H1: stabilizes the DNA between the nucleosome cores.

D2 Chromatin Structure D2 Chromatin Structure — — Linker DNALinker DNA

• The additional DNA required to make up the 200 bp nucleosomal repeat, ~55 bp

D2 Chromatin Structure D2 Chromatin Structure — — The 30 nm fiberThe 30 nm fiber

•Higher ordered•Left-handed helix•Six nucloesomes per turn

D2 Chromatin Structure D2 Chromatin Structure — — Highter order Highter order structurestructure

• On the largest scale, chromosomal DNA is organized into loops of up to 100 kb in the foem of the 30nm fiber

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — The mitotic chromosomeThe mitotic chromosome

Centromere

Telomere

Mitotic chromosome at metaphase

Nuclear matrix Loops of 30nm fiber

Sister chromatid

Chromatid

Mitotic chromosome

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — The centromereThe centromere

• The region where two chromatids are joined.• The sites of attachment to the mitotic spindle vi

a kinetochore.• Centromere DNA.

Chromosomal components:Chromosomal components:①① ChromatidChromatid②② Centromere/Primary ConstrictionCentromere/Primary Constriction③③ Short armShort arm④④ Long armLong arm

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — TelomeresTelomeres• Specialized DNA sequences which form the ends of the l

inear DNA of the eukaryotic chromosome.• Contains up to hundreds copies of a short repeated sequ

ence (5’-TTAGGG-3’ in human).• Synthesized by the enzyme telomerase (a ribonucleoprotein) independent of normal DNA replication.• The telomeric DNA forms a special secondary structure to protect the chromosomal ends from degradation.

Human chromosomes (grey) capped by telomeres (white).

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — Interphase chromosomeInterphase chromosome

• In interphase, the chromosomes adopt a much more diffuse structure, although the chromosomal loops remain attached to the nuclear matrix.

• Cannot be visualized individually.

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — Heterochromatin Heterochromatin 异染色质异染色质

• Highly condensed

• Transcriptionally inactive

• Can be the repeated satellite DNA close to the centromeres, and sometimes a whole chromosome (e.g. one X chromosome in mammals)

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — EuchromatinEuchromatin• Euchromatin: chromatin other than heterochrom

atin. • More diffused and not visible• The region where transcription takes place• Not homogenous, only a portion (~10%) euchro

matin is transcriptionally active where the 30nm fiber has been dissociated to “beads on a string” structure and parts of these regions may be depleted of nucleosome.

•The nucleus of a human cell showing the location of heterochromatin.

Animal cells

Plant cells

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — DNase hypersensitivityⅠDNase hypersensitivityⅠ

• Active regions of chromatin, or regions where the 30nm fiber is interrupted by the binding of a specific protein to the DNA, or by ongoing transcription, are characterized by hypersensitivity to deoxyribonuclease (DNaⅠse ).Ⅰ

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — CpG methylationCpG methylation• Methylation of C-5 in the cytosine （胞嘧啶） base of 5’-

CG-3’• Occurs in mammalian cells• Signaling the appropriate level of chromosomal packing

at the sites of expressed genes • CpG methylation is associated with transcriptionally inac

tive regions of chromatin• Islands of unmethylated CpG are coincident with region

s of DNase I hypersensitivity• “Islands”: surround the promoters of housekeeping gene

s.• Responsible for epigenetic （渐成说） and may also to

RNA silencing.

D3 Eukaryotic Chromosomal Structure D3 Eukaryotic Chromosomal Structure — — Histone variants and modificationHistone variants and modification

• The major mechanisms for the condensing and decondensing of chromatin operate directly through the histone proteins which carry out the packaging.

• Histones undergo posttranslational modifications which alter their interaction with DNA and nuclear proteins.

• The H3 and H4 histones have long tails can be covalently modified. Modifications of the tail include methylation, acetylation, phosphorylation, etc.

• The core of the histones (H2A and H3) can also be modified. • Histone modifications act in diverse biological processes such

as gene regulation, DNA repair and chromosome condensation (mitosis).

D4 Genome complexityD4 Genome complexity — — Noncoding DNANoncoding DNA

• DNA sequence that does not code for protein or RNA, including

• Introns (unique sequence) in genes

• DNA consisting of multiple repeats, can be tandemly repeated sequences (e.g. satellite DNA) or interspersed repeats (e.g. Alu element) etc.

D4 Genome complexityD4 Genome complexity — — Reasociation KineticsReasociation Kinetics （复性动力（复性动力学）学）

D4 Genome complexityD4 Genome complexity — — Unique sequence DNAUnique sequence DNA

• The slowest to reassociate （复性最慢）• Corresponds to coding regions of genes o

ccurring in one or a few copies/haploid genome

• All the DNA in E. coli genome has a unique sequence.

D4 Genome complexityD4 Genome complexity — — Tandem gene clustersTandem gene clusters

• Tandem gene clusters:

(1) moderately repetitive DNA consists of a number of types of repeated sequence.

(2) genes whose products are required in unusually large quantities, e.g. there are 10-10000 copies of rDNA encoding 45S precursor and X100 copies of histone genes.

D4 Genome complexityD4 Genome complexity — — Dispersed repetitive DNADispersed repetitive DNA

• Moderately repetitive (x100- x1000 copies)

• Scattered throughout the genome

• Human Alu elements: 300bp, 300 000 –500 000 copies of 80-90% identity

• Human L1 element

• Alu + L1= ~ 10% of human genome.

• Functions of these repetitive DNA ： largely unknown

D4 Genome complexityD4 Genome complexity — — Satellite DNASatellite DNA• Highly repetitive DNA (>106), very short (2 to 20-30bp,

mini- or micro-), in tandem arrays• concentrated near the centromeres and forms a large p

art of heterochromatin.• as separate band in buoyant density gradient• no function found, except a possible role in kinetochore

binding• Minisatellite repeats are the basis of the DNA fingerprin

ting techniques.

D4 Genome complexityD4 Genome complexity — — Genetic polymorphismGenetic polymorphism

1. Single-nucleotide polymorphism

4. Single strand conformation polymorphism

3. Restriction fragment length polymorphism

2. Simple sequence length polymorphism

D5 The flow of genetic informationD5 The flow of genetic information — — The central dogmaThe central dogma

D5 The flow of genetic informationD5 The flow of genetic information — — Prokaryotic gene expressionProkaryotic gene expression

5‘

3‘ 5‘

DNADNA 3‘Transcribed regionPromoter Terminator

5‘ pop

mRNmRNAA OH 3‘

AUG

RBS

stop AUG stopRBS

TranscriptiTranscriptionon

RNA RNA polymerasepolymerase

ProteinsProteins

Ribosomes, aminoaRibosomes, aminoacyl-tRNAscyl-tRNAs

TranslatiTranslationon

D5 The flow of genetic informationD5 The flow of genetic information — — Eukaryotic gene expressionEukaryotic gene expression

5‘

3‘ 5‘

DNADNA 3‘Transcribed regionPromoter

5‘ pop

Pre-mRNAPre-mRNA AUG

Poly( A) siteINTRON

stopEXONS

TranscriptiTranscriptionon

RNA polymerase ⅡRNA polymerase Ⅱ

mRNmRNAA

Splicing, capping, pSplicing, capping, polyadenylationolyadenylation

RNA RNA processingprocessing

5‘ MeGppp

AUG

Poly( A) tail

stop

Ribosomes, aminoaRibosomes, aminoacyl-tRNAscyl-tRNAs

TranslatioTranslationn

CAPAAA…3‘

ProteinProteinss

Cytoplasm－－－ cellular organelles

Mitochondrion

类囊体

Chloroplast

The endoplasmic reticulum (ER) is responsible for the production of the protein and lipid components of most of the cell's organelles.

池

Golgi Apparatus 。 The Golgi is principally responsible for directing molecular traffic in the cell

Cellular organelles you should further understand

Cellular organelles（continue）

Lysomoes（溶酶体），which contains hydrolytic enzymes

肌动蛋白

Cytoskeleton(细胞骨架 )

The ribosome plays a key role in the synthesis of proteins.

The vacuole is used only in plant cells. It is responsible for maintaining the shape and structure of the cell.

Multiple choice Multiple choice questionsquestions

1 ． Which of the following is common to both E. coli and eukaryotic chromosomes?

A the DNA is circular. B the DNA is packaged into nucleosomes.C the DNA is contained in the nucleus. D the DNA is negatively supercoiled. 2 ． A complex of 166 bp of DNA with the histone octa

mer plus histone HI is known as a . . . A nucleosome core. B solenoid. C 30 nm fiber. D chromatosome.

3 ． In what region of the interphase chromosome does transcription take place?

A the telomere. B the centromere. C euchromatin. D heterochromatin. 4 ． Which statement about CpG islands and methylation is not tr

ue? A CpG islands are particularly resistant to DNase I. B CpG methylation is responsible for the mutation of CpG to TpG in e

ukaryotes. C CpG islands occur around the promoters of active genes. D CpG methylation is associated with inactive chromatin.

5 ． Which of the following is an example of highly-repetitive DNA?

A Alu element.

B histone gene cluster.

C DNA minisatellites.

D dispersed repetitive DNA.

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