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The cell NucleusThe cell NucleusThe cell NucleusThe cell Nucleus

XiamixinuerXiamixinuer··Yilike Yilike

Chapter 9

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Chapter 9 Nucleus

Learning ObjectivesLearning Objectives1. Mastering: ultrastructure of nuclear envelop; 1. Mastering: ultrastructure of nuclear envelop;

nuclear pore complex; composition and four levels nuclear pore complex; composition and four levels organization of chromatin; packaging of chromatin; organization of chromatin; packaging of chromatin; types of chromatin.types of chromatin.

2. Comprehending: structure and function of 2. Comprehending: structure and function of nucleolus; function of nuclear pore complex; nucleolus; function of nuclear pore complex; process of RNA processing.process of RNA processing.

3. Understanding: basic function of nucleus; nuclear 3. Understanding: basic function of nucleus; nuclear matrix.matrix.

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1. The nucleus: Nuclear envelope and NPC

A. Structure: Double-membrane nuclear envelope surrounds the nucleus

Structure of the interphase nucleus

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The main functions of nucleusThe main functions of nucleus

1. Carry genetic information （ DNA ） ;

2.Duplicate,transcript of genetic information and control protein synthesis;

3.Regulation and control centre of living action of cells.

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NP(nucleoplasmic index)

NP= Vn/(Vc － Vn)

Usually size of nucleus can be estimated by calculate the NP.

Normal cell NP≈0.5， Dividing cell NP>0.5， Aging cell NP<0.5。

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A typical nonmitotic nucleus includes four major components.

nuclear envelope

nucleoluos

chromatin

matrix

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B. The nuclear envelope consists of two membranes by a perinuclear space.

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The inner surface of the nuclear envelope is lined by the nuclear lamina

The nuclear lamina supports the nuclear envelope: Gives shape and stability of nuclear envelope;

Provides a structure link between chromatin and nuclear envelope;

The nuclear lamina is composed of lamins.

The integrity of the nuclear lamina is regulated by phosphorylation and dephosphorylation.

12Breakdown and reformation of nuclear envelope during mitosis

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The structures of nuclear envelope

outer nuclear membrane

inner nuclear membrane

perinuclear space

nuclear pores

nuclear lamina

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C. Nuclear pore complex (NPC)

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The structure of

Nuclear pore

complex (NPC)

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Old model structure

Ring subunit： 8 pair

Annular subunit： 8

Central plug ： 1

fibril：

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nuclear lamina

pore蛋outer membrane

Inner membrane

Perinuclear space

A C A CA CA CA C A C A C

B B B B B B B lamina A.B.C

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D. Molecules enter and exit the nucleus through nuclear pore complex

Bidirectional traffic

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Molecules enter and exit the nucleus through nuclear pore complex

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Passive transport—passively diffuse

3000-4000 NPC/cell(mammalian);

To import about 106 histone/3 mins.(DNA-sythesizing cell) = 100 histone/ min/NPC

Each NPC contains one or more open aqueous channels: 9 nm in diameter and 15 nm long

The effective size of these channels has been determined by injecting various sizes of colloidal gold particles and examined by electron microscopy.

<10 nm in diameter

<60kd globular proteinAble to enter the nucleus

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Active transport

Transport of large proteins into nucleus needs nuclear localization signal (NLS)

Pore diameter is about 9nm but much larger objects, including ribosomal subunits can pass through (albeit slower than smaller molecules). This implies that recognition of appropriate signal allows temporary stretching of the pore.

Energy is required.

22Transport of mRNA out of the nucleus

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Nucleolus and ChromosomesChapter 9 section 2

Learning Objectives

(1) The components of chromatin and packaging of

chromosome

(2) Nucleolus.

24human nucleus under LM

human lymphocyte nucleus under SEM

Chromatin fiber

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Each human cell contains about 2 m of DNA within nucleus if stretched end-to-end, yet the nucleus of a human cell itself is only about 6 μm in diameter.

Compaction ratio=nearly 10000-fold. (Chromosome 22: DNA 1.5cm2 μ m)

Eukaryotes package DNA in Chromatin and chromosomes

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Chromosomes exist in different states throughout the life of a cell.

Chromatin: (Interphase)

Fibers, 10-30nm in diameter,dispersed through the nucleus.

DNA+Proteins+non-Proteins+RNA.

Chromosomes: (M phase)

Cell division, these fibers condense and fold into larger, compact structure.

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Levels of organizatio

n of

chromatin

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B. Nucleosomes are packed together to form chromatin fibers and chromosomes

4 degree folding model4 degree folding model Nonhistone proteins provide a

structural scaffold for long chromatin loops.

2nm DNA –11nm nucleosome – 30nm fiber – Loop– Metaphase chromosome

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4 degree folding model4 degree folding model

First degree:

Nucleosomes Second degree:

Filament 10nm in diameter Third level:

Fiber,30nm in diameter Fourth level:

Chromosome

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A. DNA packaging:First degree of packaging is Nucleosomes

Nucleosomes are the basic unit of chromatin structure.

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Evidence:

(1)Electron micrographs of chromatin fibers

Isolated from interphase nucleus: 30nm thick

Chromatin unpacked, show the nuclesome

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Evidence:

(3)X-ray diffraction studies

3-D of nucleosome Nature 389:251, 1997

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Structure of nucleosome

Digested briefly:

H1+Octamer+200bpDNA

Digested longer:

Octamer+146bp

H1 is released

Core particle

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Structural Structural

organization of organization of

the nucleosomethe nucleosome

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CORE OF PERIPHERAL HISTON

H4

DNA (146bp 、 1.75 ROUND）

H3

H4H3

H2AH2A

H2B

H2B

H4H3

H3H4

H2AH2A

H2B

H2B

10nm

LINKER DNA （ 60b

p）

H1

H1

NUCLEOSOME

CORE

LINKER

DNA MOLECULE ：146bp 、 1.75 ROUND

HISTONE ：2 （ H2A 、 H2B 、 H3 、 H4 ） OCTAMER

HISTONE ： H1

DNA MOLECULE ： 60bp

NUCLEOSOME

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A histone octamer forms the nucleosome core

Histone octamer:

(H2A-H2B)-(H3-H4)-(H3-H4)-(H2A-H2B)

Where is the histone H1?

H1 molecules are associated with the linker region. 146+15~50bp linker DNA

200bpDNA:

Linker DNA:15-50bp

Nucleosomal DNA:146bp to wrap 1.75 times around the histone core.

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Evidence:

(2)Nuclease digestion (Rat liver chromatin)

The basic repeat unit, containing an average of 200bp of DNA associated with a protein particle, is the nucleosome

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Histones:

The most abundant proteins associated with eukaryotic DNA

Rich in positively charged basic amino acids, which interact with the negatively charged phosphate groups in DNA

The amino acid sequences of histones H2A, H2B, H3, H4 are remarkably similar among distantly species

HistoneNumber of Residues

Mass(KDa

)Arg% Lys%

H1 215 23.0 1 29

H2A 129 14.0 9 11

H2B 125 13.8 6 16

H3 135 15.3 13 10

H4 102 11.3 14 11

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Second level: nucleosome filament10nm in diameter

H1

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Third level:

fiber,30nm in diameter

Fourth level: chromosome

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Summary of 4 degree folding model

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B. Euchromatin

and

Heterochromatin

Euchromatin

The possibility of transcription;

The types of chromosomal structure—30-nm fibers and looped domains;

Light-staining, less condensed;

Transcriptional activity

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Heterochromatin:

Dark-staining, condensed chromatin;

No transcriptional activity; in a typical

mammalian cell, approximately 10% of

the genome is packaged into

heterochromatins forming CEN and TEL

Divided into two classes:

Constitutive & facultative

Compacted state at all time: Centromere

Inactivated at certain phase of life

Example of facultative heterochromatin:

Random inactivation of X chromosome in different cells during early embryonic development

Barr body in a woman’s cell

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3. Chromosome number,size,and shape at metaphase are species specific

ChromatidsKaryotype Banding

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Human mitotic chromosomes and karyotype

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C. Main structures of chromosome

Centromere & Kinetochore

Centromere: Highly repeated DNA+Kinetochore

The Centromere and Kinetochore: serve as a site for the attachment of spindle microtubules during mitosis and meiosis

The structure of a human CEN

Main structures of chromosome: Including centromere, arm (p and q), secondary constriction, telomere and satellite.

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secondary constriction

nucleolar organizing region,NOR

satellite

telomere

centromere

A typical mitotic chromosome at metaphase

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Three functional elements are required for replication and stable inheritance of chromosomes

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Functions

Protect the chromosomes from nuclease influence

For the complete replication of chromosome

Prevent the ends of chromosomes from fusing

Telomeres:

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Telomere, telomerase and cellular aging, cancer cell

Telomerase is found in germ cells, not in somatic cells.

The telomere length of adult is shorter than that of younger.

Telomere shortening is thought to activate a suicide program.

So, telomere shortening plays a key role in protecting the body from cancer.

90% of human tumors contain an active telomerase.

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D. Nucleolus a. Structure

Fibrillar centers

Dense fibrillar component

Granular component

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b. Functions of nucleolus: Ribosomal Biogenesis

Ribosomal Biogenesis:Ribosomal Biogenesis:

pre-rRNA synthesispre-rRNA synthesis ProcessProcess AssemblyAssembly

FC. DFC DFC.GC Cytosol

Directional processDirectional process

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Characteristics of RNA transcription

NORs in human chromosomes:

13\14\15\21\22

Code for (in eukaryotes):

18s, 28s, 5.8s rRNA

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rRNA genes are tandemly arrayed in genome: Christmas-Like tree when transcripted

Primary transcripts condense immediately with a number of proteins to form particles about twice the size of nucleosomes. These particles are called hnRNP (heterogeneous nuclear ribonucleoprotein) particles.

These likely associate into “spliceosomes” involved in processing

Also found in the nucleus are snRNPS (small nuclear ribonucleoproteins). These are believed to also contribute to RNA splicing and to histone mRNA synthesis

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Cleavage of pre-rRNA as RNP particles

Mammalia

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Synthesis and processing of 5s rRNA

5s rRNAs are encoded by a large number genes (Human, 2000)

5s rRNA gene are located outside the nucleolus.

5s rRNAs are transcibed by RNA poly III.

The 3’ end of 5s rRNA is removed during processing.

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The assembly of ribosomes

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The nucleolus disappears during mitosis

Nucleolar fusion of human fibroblast in culture

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Summary

(1). The components of the typical nonmitotic nucleus

(2). The components of chromatin and packaging of chromosome: Scaffold radial loop structure model.

(3). Nucleolus components and functions.

Homework

Nucleolus Structure includes which parts? What are the functions of nucleolus? Describe briefly the difference between

euchromatin and heterochromatin. Describe briefly structure of nucleosome? Any Questions??

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