II. INTERNAL ORGANIZATION OF EUKARYOTIC CELLS

Structure of a „typical” animal cell

Structure of a „typical” plant cell

II.1.4. Nucleus

Biological significance: storage, expression and transmission of the genetic information.

Segmented nuclei of leukocytesSegmented nuclei of leukocytes

neutrophils

Structural components

II.1.4.1. Nuclear membrane (envelope)

cytoplasm

nuclear membrane nuclear lamina

chromatin

nuclear pore

Biological significance: limiting layer between the inner part of the nucleus and the cytoplasm. Specific transport of micro- and macromolecules.

The nuclear envelopenuclear envelope is formed from two concentric membranes that are continuous with the endoplasmic reticulum (ER). The space between the two membranes is the perinuclear spaceperinuclear space which is continuous with the lumen of the ER.

The inner nuclear membrane contains proteins which enable it to bind to the nuclear laminanuclear lamina (network of intermediate filaments), which binds the chromatin.

The double-membrane envelope is penetrated by nuclear poresnuclear pores.

Nuclear pore

EM imageConstruction

surface wiev

side wiev

Relationship of the nucleus and the cytosol

II.1.4.2. Chromatin

The chromatin is composed of deoxyribonucleic acids and proteins. In interphase they are arranged in a relatively loose , „network” form. EM shows that there are two different forms of the chromatin, one appearing as a light element (euchromatin), the other one being dark (heterochromatin).

HeterochromatinHeterochromatin: Closely packed chromatin . The DNA is organized by histon and non-histone proteins.

Euchromatin:Euchromatin:

Here the chromatin is loosely arranged. The DNA double helices are partially split to single strands by RNA polymerases.

Euchromatic nucleus of neuronEuchromatic nucleus of neuron

H2A, H2B, H3 and H4 (two molecules of each) are building the Histone-Octamer . The DNA double helix makes two turns around this octamer (166 base pairs). H1 binds to theis complex.

(packing level: 6-7x) H1

Octamer

1. Level of organisation: NucleosomeNucleosome

Structure

The chromatin is a DNA-protein complex About half of the chromatin is protein: they are either histones or non-histone proteins.

Histones Histones are small, positively charged proteins, which bind non-specifically to DNA end compensate the negative charge of phosphate groups of the DNA.

Histone classes: H1, H2A, H2B, H3 and H4.

2. Level of organisation : 30 nm chromatin fiber30 nm chromatin fiber

The nucleosomes are spirally organized around the H1 core (packing level: ~ 40 x)

This structure is broken to segments by binding of non-histone proteins (e.g. gene regulator proteins).

3. Level of organisation: 300 nm chromatin fibers300 nm chromatin fibers

30 nm chromatin fiber

loop domains

20 000-30 000 base pairs

300

nm

Chromosomes

W. Waldeyer, 1888:

" …thick chromatin structures which can be seen during mitosis and meiosis...„

Name: chroma: color and soma: body

Boveri and Sutton, 1905. Chromosome theory: the chromoses are the carriers of the genetic information..

Chromosomes exists only during cell division as microscopically Chromosomes exists only during cell division as microscopically detecteble units. detecteble units.

At the start of the mitosis (M phase) each chromose has replicated and consists of two sister chromatids joined together

Fine structure

Chromatids: 300 nm chromatin fiber form spirals (packing level: 10 000 x)

300 nm

700 nm

Staining of the chromosomes

• Fluorescent stains (e.g. Quinacrin): Q-Q-bands bands specific for AT-rich DNA regions

• Giemsa-staining:according to the technique applied, either G-bandsG-bands ("Giemsa-positiv")

specific for AT-rich DNA-regions or

R-bandsR-bands ("Giemsa-negativ")

specific for GC-rich DNA-regions

ChromosomesThe human karyotype

fruitfly 8 house fly 12 ant 48 frog 26 carp 104 dog 38 cat 64 mouse

40 rat42

cattle 60 rhesus monkey 42 human 46

baker’s yeast 32 corn

20 rice 24 wheat

42 tobacco 48

Chromosomal organization of different species

Variability in chromosomal organization.

Chromosomal organization (karyotype) can show high variability even in closely related species.

The number of genes is equal!

Reeves deer

Indian deer

Scanning-EM, human

chromosomes

Schematic view of

chromosomal regions

X 4 5

Tel omer

shorter arm „p”

longer arm „q”

centromer1

23

45

1

1 1

111

2

2

2 233

sister chromatids

Structure of the chromosomes

Telomers, aging, cancer (and Dolly)

Human telomer: 15 000 base pairs of repeated TTAGGG DNA sequences. Shortening: 50-200 base pairs/division.

In germline cells: telomere terminal transferase (telomerase): ribonucleoprotein reverse transcriptase.

Binding of the chromosomes to the mitotic spindle

Attachment site:

kinetochorkinetochor at the centromer

Preparation of chromosomes, karyotype determination

Blood culture (phytohaemagglutinin) colchicin separation of erythrocytes and white blood cells fixation staining

microphotoarrange according to size, centromer position and banding pattern