Nucleus And Chromosomes

Nucleus and chromosomes Xu Zhaoyang Department of cell biology Basic Medical college Zhengzhou university

A brief summary to nucleus The cell nucleus is a remarkable organelle becauseit forms the package for our genes and their controlling factors. Cell's control centre (usually only 1) Often spherical, 5-10 microns diameter large and pale in active cells

Organization of the nucleus Nuclear envelope Nuclear matrix Nucleolus Chromatin nulceoplasm

The nuclear envelope The nuclear envelope is shown in an electron micrograph in the figure to the right. The filaments outside the envelope are not visualized with these protocols. Also, the nuclear lamina just inside the nuclear envelope is not shown well. However, one can see ribosomes on the outer membrane and the sac enclosed by the two membranes.

The envelope of nucleus The space between the outer and inner membranes is also continuous with rough endoplasmic reticulum space. The inner nuclear membrane (above)defines the nucleus itself. The outer membrane (right) is continuous with the rough endoplasmic reticulum and has ribosomes attached.

Nuclear envelope The nuclear envelope has two membranes, each with the typical unit membrane structure. They enclose a flattened sac and are connected at the nuclear pore sites. The nuclear envelope is enmeshed in a network of nuclear lamina for stability.

Selective transport materials to and from the nucleus Diffusion 5,000 MW are freely diffusable 17,000 MW-- take 2 min to establish equilibrium 44,000 MW--take 30 min to establish equilibrium 60,000 MW--cannot move in by diffusion

Active Transport This form of transport is assumed when molecules larger than the pore diameter (10 nm) get into the nucleus. Studies with gold markers show that the pore can actually dilate up to 26 nm when it gets the appropriate signal.

The nucleolus The nucleolus is the most obvious structure seen in the nucleus when viewed in the light microscope. The nucleolus is organized from the "nucleolar organizing regions" on different chromosomes. A number of chromosomes get together and transcribe ribosomal RNA at this site.

The structure of nucleolus The nuclear organizing (NO) regions are seen as circular areas (pale) surrounded by a rim of electron dense filaments. These filaments collectively are called the pars fibrosa (PF). This is formed from newly transcribed ribosomal RNA. After the ribosomal RNA is transcribed, it is linked to proteins and one can see accumulation of ribonucleoprotein particles in the pars granulosa (PG). These particles form the two types of ribosomal subunits (large and small) which are then transported out of the Nuclear Pores separately.

The nucleolus A preparation of nucleoli is dissociated and spread on a liquid surface. Then, the synthesis of ribosomal RNA is stimulated and after a period of time, the DNA from the nucleolar organizing region begins to look like a Christmas tree. The top of the tree is the start site. As you move down the "tree", the branches appear longer. Each branch is a growing strand of ribosomal RNA. The DNA code is being transcribed and the nucleotides added to the growing RNA strand.

The function of nucleolus rDNA 45S rRNA 32S rRNA

18S rRNA protein

5S rRNA5.8S

Small subunit Transcribe from the DNA out of the nucleolus

rRNA 28S rRNA

protein

large subunit

The nuclear matrix The interphase nucleus is thought to contain a 3-dimensional filamentous protein network referred to as nuclear matrix. It can be isolated by dissolving nuclei in lipid detergents and solubilizing most of the DNA with DNases.

The nuclear matrix Transmission electron micrograph (X47,000) of a portion of nuclear matrix (left) and surrounding cytoplasm. Cytoskeletal filaments are clearly visible.

The function of matrix It provides a framework to maintain the overall size and shape of the nucleus. The matrix acts as a structural attachment site for the DNA loops during the interphase: evolutionary highly conserved 300-1000 bp long DNA sequences, referred to as MAR (Matric-attached Region), have been identified that define the base of DNA loops, anchoring them to specific proteins. By means of such chromosomal attachment sites, the matrix might help to organize chromosomes, localize genes, and regulate DNA transcription and replication within the nucleus.

Speculative model of interphase chromatin organization, envisioning the nuclear matrix as a series in internal channels . Chromosomal DNA loops attached to the nuclear matrix through replication origins.

Chromatin and chromosome Chromatin and chromosome are the same material with different structure in the different stage of cell division. The chromatin is composed of DNA, histone(H1 H2A H2B H3 H4 ) ,non-histone (help to coiling chromatin into chromosome, ravel the DNA suppressed by histone) and RNA.

Two kinds of chromatin Heterochromatin

Euchromatin

Heterochromatin Heterochromatin is the condensed form of chromatin organization. It is seen as dense patches of chromatin. Sometimes it lines the nuclear membrane, however, it is broken by clear areas at the pores so that transport is allowed. Sometimes, the heterochromatin forms a "cartwheel" pattern. Abundant heterochromatin is seen in resting, or reserve cells such as small lymphocytes (memory cells) waiting for exposure to a foreign antigen. Heterochromatin is considered transcriptionally inactive.

Euchromatin Euchromatin is threadlike, delicate. It is most abundant in active, transcribing cells. Thus, the presence of euchromatin is significant because the regions of DNA to be transcribed or duplicated must uncoil before the genetic code can be read.

Nucleosomes: the lowest level of chromosome organization The nucleus is only 6 micrometers in diameter. The total length of DNA in the human genome is 1.8 meters. Thus, in order to pack the DNA into the nucleus as in the photograph of the metaphase chromosome , there must be several levels of coiling and supercoiling. There is nearly a 10,000-fold reduction in length in an interphase nucleus. Each chromosome contains 1 long molecule of DNA plus associated histones (basic proteins) which help in the condensation and regulation processes.

Nuclearsome histone octomer + DNA = nucleosome 146 nuceotides + 2H2a + 2H2b + 2H3 + 2H4 (histone octomer) nucleosomes joined by linker DNA and histone H1 to form chromatin fiber .

The nuclearsomes in Electron microscope A length of chromatin that has been experimentally unpacked ,or decondensed,after isolation to show nucleosomes

Solenoid Model of Chromosome Structure first level : nuclearsome (packing ratio -7) 2nd level: 6 nulearsomes coiling into a helical array - 30nm solenoid (packaging ratio -6) 3rd level : Solenoid coiling into a 400nm supersolenoid (packaging ratio -40) 4th level: chromotids (packaging ratio -5)

Scaffold-radial loop structure model

The function of nuclear Store genes on chromosomes; Organize chromatin into chromosomes to allow cell division; Transport gene products via nuclear pores; Produce messages (mRNA) that code for proteins; Produce ribosomes in the nucleolus; Organize the uncoiling of DNA to replicate key genes;

Nuclear pore complex An electron micrograph of a nuclear pore. It appears as if the two membranes are pinched at that site, leaving a space filled with filamentous material. Sometimes a thin diaphragm may be seen running horizontally through the pore

The nuclear pore This electron micrograph shown in the figure to the right depicts a nuclear pore complex seen with the transmission electron microscope. As is obvious, little detail can be seen. The inner and outer membranes of the nuclear envelope are joined and there appears to be a diaphragm-like structure in the center. However, the intricate detail pictured in the foregoing figure cannot be appreciated.

The structure of the pore complex The right figure shows a view of the nuclear pore from the top. It contains 8 subunits that "clamp" over region of the inner and outer membrane where they join. Actually, they form a ring of subunits 15-20 nm in diameter. Each subunit projects a spoke-like unit into the center so that the pore looks like a wheel with 8 spokes from the top. Inside is a central "plug". The next (left) figure shows a cross section of the pore with the clamp-like complex adjacent to the membranes. The projected spoke is directed towards the central "plug' or granule.

The method to visualize the nuclear pore complex Negative staining This protocol deposits heavy metal stains around structures and delineates their surface structure. When placed in an electron microscope, the heavy metal around the structure retards the electron beam. The structure itself allows the electron beam to pass and this activates the photographic emulsion. Thus, a "negative" image is created in the photograph.

Freeze-fracture/freeze-etch This protocol involves the rapid freezing of structures followed by fracturing. The membranes are cleaved along their lipid bilayer and either the face next to the cytoplasm (protoplasmic or P face) or the extracellular (E) face of the membrane is shown. Then, a replica is made of the membrane by evaporating heavy metal over the surface. This replica is what is viewed in the transmission electron microscope

Negative stainning The figure to the right illustrates a preparation of nuclear pore complexes that were isolated from an oocyte and spread on plastic. Then, the heavy metal stain was applied to delineate their structure. Note that one can visualize the 8 subunits, the spokes of the wheel and the central granule.

Freeze-fracture/freeze-etch The right figure shows a surface view of nuclear pores scattered in the inner nuclear envelope membrane. The subunits cannot be appreciated with this preparation. However, it can be used to study formation of nuclear pore complexes. This varies with the physiological state of the cell.

Another preparation The subunits forming the rings and their spokes. Note that one of the pores appears to be open in the center, forming a channel. The subunits also project fibrils from either side. At the nuclear side, these fibrils join to form a "nuclear cage" The fibrillar structures cannot be appreciated in this micrographs.

Nuclear lamina An electron micrograph of a portion of the lamina in a Xenopus oocyte prepared by freeze-drying and metal shadowing. The lamina is formed by a regular lattice of specialized intermediate filaments.

Structural characteristics of nuclear lamina 1. Consists of "intermediate filaments", 30-100 nm thick. 2. These intermediate filaments are polymers of lamin, ranging from 60-75 kD 3. A-type lamins are inside, next to nucleoplasm; Btype lamins are near the nuclear membrane (inner). They may bind to integral proteins inside that membrane.

Function of nuclear lamina Play a role in assemble and disassemble of envelope before and after mitosis. After they are phosphorylated, this triggers the disassembly of the lamina and causes the nuclear envelope to break up into vesicles. Dephosphorylation reverses this and allows the nucleus to reform. If antibodies to lamins are injected into cells, the nuclei cannot reform after cell division. Therefore, these lamins are vital to reassembly.

Function of nuclear lamina Provide a dock place for chromatin .Arrange the chromatin on the inner side of lamina to stop the coiling process. In prophase, accompanied by the disassemble of the laminar, the chromatin lost the anchor, coiling into chromosome.

Function of nuclear lamina

Play a role in assemble of the nuclear. Removed of the laminar will suppress the assemble of envelope and the pore complex.

Signal The signal is in the peptide sequences. These are recognition sequences rich in lysine, arginine, and proline. Signal may control direction of transport: Goldlabeled tRNA or 5S RNA may leave the nucleus, but may not come back. Also, transport of RNA is inhibited by alteration of the 3' end or the 5' cap structure.

The evidence for the signal existance A peptide sequence, called nucleoplasmin, was isolated and linked to colloidal gold. It was then injected into an oocyte and traced with electron microscopy. As shown in the figure to the right, the gold particles mark the site of transport of the nucleoplasmin and the studies showed that it was transported into the nucleus. Small gold markers are evident inside the nucleus.

The evidence that transport requires energy Transport of mRNA can be inhibited by cooling the cells (placing them at 4 C). ATP Hydrolysis is required to import a protein into the nucleus. In the absence of ATP, the proteins bind specifically at the cytosolic face of isolated frog oocyte nuclei. When ATP is added, the proteins are allowed to enter. This can be traced with colloidal gold labeling of the proteins. Studies show that ATP is needed for entry, but not for binding to specific receptors.