Cell nucleus, chromatin, chromosome. Replication.

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Cell nucleus, chromatin, chromosome. Replication. by Krisztina H.-Minkó Semmelweis University Department of Human Morphology and Developmental Biology 14. 09. 2015.

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

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

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

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

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

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

The cell nucleus contains chromatin

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

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

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)

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

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

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.

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

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.

dER Nuclear membrane Nucleus

Nuclear pore dER Nuclear membrane nucleus

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

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

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.

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’

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.

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.

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.

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 10.000x. 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

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

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)

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

DNA and RNA transcription video - real time http://www.youtube.com/watch?v=TSv-Rq5C3K8

References Textbook: Essential cell biology, p. 56-58, 74-75, 172-192, 184-191, 198-210, 232-244, 502-504, 576-577 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