1. Cell nucleus, chromatin, chromosome. Replication.
by Krisztina H.-Minkó
Semmelweis University
Department of Human Morphology and Developmental Biology

2. Only eukaryotes have cell nucleus
Prokaryote (archea, bacteria): no cell nucleus
Organisation of the genetic material is more simple
Eukaryote – cell nucleus
The cell nucleus mesure 5-10 µm
in most of the cells

3. Figure 1-29 Essential Cell Biology (© Garland Science 2010)

4. bacteria (prokaryote)
Makrophag (eukaryote) with phagocytosed bacteria (prokaryote)
bacteria (prokaryote)
makrophag (eukaryote)

5. Light microscopic appearance of the nucleus
Spinal cord – Nissl staining
Epididymis – Haematoxylin-Eosin staining
Blood smear
May-Grünwald-Giemsa staining
Hyalin cartilage – Toluidin blue staining

6. Giant cells with many cell nuclei
Skeletal muscle – HE staining
Giant cells arise:
* either by fusion of individual cells: Syncytium (e.g. the striated muscle fiber)
* or by mitotic cell division without division of the cytoplasm (cytoninesis): Plasmodium.
Osteoclast, bone – HE staining

7. The cell nucleus is a compartment of eukaryotic cells
Nuclear membrane
Cell nucleus
Rough endoplasmic reticulum
Rajz a táblára hogyan jöhet létre
Ribosome
Smooth endoplasmic reticulum

8. The cell nucleus contains chromatin

9. Electron micrograph (liver cell) Phase contrast micrograph
Components of the nucleus
Chromatin: basophilic substance in the nucleus, with a loose or densely packed structure (euchromatin and heterochromatin, resp.). Main molecular components: DNA and proteins.
Nucleolus: 1 or more dense, basophilic structures, main molecular component: RNA (rRNA and ribosomal subunits in assembly).
Nuclear envelope (nuclear membrane): thin envelope of the nucleus (a thin border between nucleus and cytoplasm in the light microscope, a flattened cisterna with pores in the electron microscope).
heterochromatin
nucleolus
euchromatin
cell nucleus
nuclear envelope
nuclear envelope  
Electron micrograph (liver cell)
Phase contrast micrograph

11. Different appearance of chromatin by light microscopy during different stages of the cell cycle
Interphase
During cell division
Euchromatin
Moderate condensation of the DNA, intense transcription
Heterochromatin
Intense condensation of the DNA, less transcription
Chromosomes
Maximally condensed DNA, no transcription

12. Nucleolus
The largest basophilic structure in the eukaryotic nucleus. Stains by RNA specific dyes. This is the site of ribosome synthesis and assembly („ribosome factory”). Place of ribosomal RNA (rRNA) transcription and assembly of ribosomal subunits. Ribonucleoproteins (tRNA, SRP, …) form here as well.
Nucleolus in neuronal cell nucleus
Formation of ribosomes:
5 chromosomes contain rRNA genes in the human haploid genome in multiple copies (nucleolus organisator region: NOR). In most of the cells one large nucleolus occur where the 10 chromosomal NOR regions are directed towards one another.
First the transcribed rRNA is cut into 3 portions, another part (5,8S RNA) is transcribed from another chromosome.
Ribosomal proteins are imported by the nuclear pores from the cytosol and assembly with the rRNAs in the nucleolus.
The created ribosomal subunits are exported by the pores into the cytosol.
Granular component (ribonucleoproteiin particles)
Filamentous component (RNA)

13. Neuronal nucleus, EM-picture
Nucleolus II.
Number of nucleoli: 1 in interphase, many before and after cell division (corresponding to NOR chromosomes)
Intense protein synthesis:
Big nucleolus, basopil cytoplasm
Neuronal nucleus, EM-picture
liver– HE staining

14. Nuclear membrane Perinuclear cistern Ribosome Outer membrane
Inner membrane
Pore complex
Nuclear lamina

15. Nuclear lamina Prometaphase Telophase
Nuclear lamina is associated to the inner nuclear membrane and is a dense fibrillar network composed of intermediate filaments (lamins) and membrane associated proteins. Stabilises the nuclear membrane and regulates the degradation and formation of it. During cell divison lamins are phosphorilated by enzimes in prometaphase which leads to conformational changes and finally the nuclear lamina and the nuclear membrane degrades beacause of the dissociation of the phosphorylated lamins. At the end of the cell divison (telophase) lamins are dephosphorylated and the nuclear lamina reforms with the nuclear membrane surrounding it.

16. Structure of the pore complex
The pore complexes consist of 8 peripheral units and a central canal (EM, isolated and negatively stained nuclear envelope)
Structure of the pore complex
fibril
perinuclear cistern
nuclear lamina
anular subunit
central canal
fibril
peripheral complex
perinuclear cistern
luminal subunit
„basket”
Number: several hundreds upto several thousands per nucleus
Diameter: 50 nm, central canal 10 (-25) nm
Total mass: 125 Million Da, consisting of 30 different protein species
Pore complexes seen from the inner surface of the nuclear envelope. Scanning EM

17. Transport through the nuclear pores
Proteins are synthesized in the cytoplasm, however, the tools for their synthesis (mRNA, tRNA, rRNA) are transcribed in the nucleus and must be exported into the cytoplasm. Proteins play important roles also in the nucleus and must be imported into the nucleus from the site of their synthesis, the cytoplasm. Therefore, transport through the pores in both directions are necessary.
Import of nuclear proteins from the cytoplasm into the cell nucleus (nuclear proteins are necessary for replication, transcription, regulation of gene transcription, RNA processing, DNA condensation, binding to mRNA, etc.
Export of RNA from the nucleus into the cytoplasm (mRNA, rRNA with its bound proteins, tRNA, etc.)
Molecules above the molecular mass of 50 kD are transported by a special mechanism. Speed of the transport: 500 molecules/sec, in both directions!
Nuclear import signals: short amino acid sequences on the proteins
Nuclear import signal defective, the protein remains in the cytoplasm
Nuclear import signal is intact, the protein is localised in the nucleus
An example for the import mechanism. The nuclear import signal on the protein is recognized by an importreceptor protein (importin), the protein (together with the receptor) is transported through the central canal of the pore complex with the aid of RanGDP protein into the nucleus, where GDP is exchanged for GTP, and the imported protein is released . The receptor, together with the Ran protein returns through the pore complex into the cytoplasm.

18. dER
Nuclear membrane
Nucleus

19. Nuclear pore
dER
Nuclear membrane
nucleus

20. Freeze-fractured specimen, EM.
Nuclear pores, EM  
Freeze-fractured specimen, EM.
Pore complexes, EM

21. Genom, chromatin, chromosome
Inside the nucleus:
Genom, chromatin, chromosome

22. Genom, chromatin, chromosome
Genom: the totality of genetic information belonging to a cell or an organism; in particular, the DNA that carries that information.
Chromatin: content of the nucleus, complex of DNA, RNA, histones, and nonhistone proteins.
Chromosome: Chromatin is organised into chromosomes especially in cells undergoing cell divison. Can be composed of one or two chromatids. Molecular composition: DNA and associated proteins. Rod –like structures sometimes in the light microscope.
Chromosome set: All the chromosomes of the cell. Human genom is stored in 23 different chromosomes. Diploid somatic cells contain two of each chromosome. Haploid gametes contains only one set of chromosomes.

24. DNA: deoxyribonucleic acid
Long, linear macromolecule, polynucleotide: Adenine, Guanine, Thymine, Cytosine. Nukleotide (pentose+ base+ phosphate group)
Double-stranded
Antiparalell structure 5’- and 3’- ends
Complementarity A-T and G-C.
Negative charging phosphate groups! 5’ 3’
Phosphate-deoxyribose
backbone 3’ 5’

25. Copying the genetic information
The genetic information (plans for the synthesis of proteins and RNAs) can be transferred onto a newly synthesized nucleic acid : DNA replication and RNA transcription.

26. Replication
DNA replication (synthesis of DNA) occurs in the cell nucleus. Synthesis of a new DNA strand along an old strand, where the base sequence in the new strand is defined by that of the old strand due to base complementarity. The two old strands are separated from each other (by helicase enzime) and serve as templates for the synthesis of the new strands. The new strand grows on its 3’ end by the addition of nucleotides, the sequence of which is determined by the nucleotide sequence in the old, complementary strand (semiconservative replication). The synthesis is carried out by the enzyme DNA polymerase. High precision!
Replication enzymes: helicase, primase, ligase, DNA-polymerase, topoisomerase
Significance of replication: DNA is replicated prior to cell division, so that the same genetic information can be sorted into the two daughter cells.

27. Chromatin structure- Proteins
Nucleosome
Histons are basic proteins (rich in Arg and Lys) (H1, H2a, H2b, H3, H4) and make ionic bonds with he DNA (which is acidic). Two of H2a, H2b, H3 and H4 histons form a central core (octamer) and DNA wound around this structure 1,5 times. Structure created by this way is called nucleosome. H1
Histon-core
(H2a, H2b, H3, H4)
DNS-double helix
Non-histon proteins also participate in chromatin condensation, gene regulation and replication.

28. DNA condensation (chromatin)
The total DNA length of a human cell is about 2m (!), while the average diameter of a nucleus is 5-10 μm. To accomodate this length of DNA in a tiny volume of the nucleus is condensation (packing) of the DNA necessary. The packing is performed by specific proteins (positively charged small proteins: histones and non-histone proteins), the end product is called chromatin. In its most condensed form (metaphase chromosomes) the DNA is shortened by about x. The chromatin in an average cell nucleus shows different levels of DNA condensation.
DNA EM
„beads-on-a-string”
„beads-on-a-string”
* nucleosome
chromatin fiber EM
chromatin fiber
loops of chromatin fiber 
metaphase chromosome  LM 
* Nucleosome: a flattened ellipsoid body composed of 8 histone proteins (core particle, 2 copies of histones H2A, H2B, H3, H4), onto which 2 DNA loops are bound.
Euchromatin
loose chromatin structure
chromatide
Metaphase chromosome

29. Different appearance of chromatin by light microscopy during different stages of the cell cycle
Interphase
During cell division
Euchromatin
Moderate condensation of the DNA, intense transcription
Heterochromatin
Intense condensation of the DNA, less transcription
Chromosomes
Maximally condensed DNA, no transcription

30. Genome organisation in interphase
A non-dividing cell contains 46 chromosomes (one chromatid in each) which means 1 DNA
molecule per chromatid.
Speicher & Carter Nature Reviews Genetics 6, 782–792 (1 October 2005)

31. Genome organisation during cell division
DNA molecules of the cell duplicate during cell division (replication). The two copies of the same DNA molecule stay together. In mitosis, condensation of the DNA takes place and metaphase chromosomes form with two chromatids containing two identical copies. Chromatids of the same chromosome are called sister chromatids (half chromosome).
Parts of the chromosome:Two "sister" Centromer is the place where cohesin proteins connect the two chromatids together.
Kinetochore proteins surround it, they serve as attachement points for kinetochore-microtubules during cell division.
Telomer: serves to protect the chromosomal ends, repetitive sequences.
Chromosomes can be identified by their length, position of the centromer and characteristic staining.
telomere
p-arm
(smaller)
DNA
centromere
q-arm
(longer)
kinetochore
chromatids

33. DNA and RNA transcription video - real time

34. References
Textbook: Essential cell biology, p , 74-75, , , , , ,
Alberts – Johnson – Lewis – Raff – Roberts – Walter: Molecular biology of the cell. 5. Auflage, Garland Science
Lectures of Dr. Pál Röhlich and Dr. Arnold Szabó
Light and EM pictures of Dep. Of Human Morphology and Developmental Biology
Neil A. Campbell, Jane B. Reece, und Jürgen Markl : Biologie, 7. Auflage Pearson Studium