Difference between revisions of "2009 Group 9 Project"

From CellBiology
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==Eukaryote Gene Expression==
 
==Eukaryote Gene Expression==
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DNA replication
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DNA in eukaryotes is formed from two strands of nucleotides, joining to give rise to the DNA double helix.  DNA replication is when an entire double stranded DNA is duplicated.  A new DNA double strand helix is formed, and is passed on to the newly divided daughter cells along with the existing parental strand.  This process is compulsory for cell reproduction prior to cell division.  The steps of DNA replication are summarized as follows:
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1. Separation of DNA double helix – The hydrogen bonds between the bases of the double strand helix are to be unwounded.  Normally this takes place at between the Adenine and Thymine bond as they are only linked by two hydrogen bonds, whereas there are three between Cytosine and Guanine.  The corresponding enzyme for this reaction is helicase.  The splitting occurs at the “origin” and the separated structure is now called the “replication fork”. Once separated, the two DNA strands act as a template for the development of the new strand.
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2. Binding of RNA Primase – The RNA primase binds to the 3’-5’ parent chain.  The function of RNA primase is to recruit RNA nucleotides (precursors for binding of DNA nucleotides), which bind to DNA nucleotides of the 3’-5’ strand.  This occurs because of the hydrogen bond attraction between the bases. 
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3. Elongation – This procedure is different for the 3’-5’ and 3’-5’ chains. 
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4. Elimination of RNA primers – RNA primers are removed by DNA PoII – exonuclease.  DNA polymerase then closes the gaps by the addition of complementary nucleotides.  The phosphate sugar backbone gap is closed by the addition of phosphate by DNA ligase.
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5. Termination -
  
 
==Variations of Nucleus in Different Cells==
 
==Variations of Nucleus in Different Cells==

Revision as of 01:01, 26 May 2009

The Nucleus

The ’nucleus’ is a double membrane organelle, arranged concentrically, in the majority of eukaryotic cells. Particular cells such as mature red blood cells and xylem may have a nucleus absent due to its loss during growth. The nucleus is predominantly seen as spherical or oval shaped in a variety of sizes. A typical mammalian cell nucleus ranges between 10-15µm and maintains genetic information in a centralised region. The nucleus is a prominent structure that denotes the difference between eukaryotic cells and prokaryotic cells by its presence.[1][2]


The double membrane of the nucleus is termed the ‘nuclear envelope’ that creates a separate compartment from the cytoplasm of a cell. This compartment contains various internal structures including chromatin, chromosomes, deoxyribonucleic acid and the nucleolus and is called the ’nuclear compartment’. The double membrane is comprised of an external membrane and internal membrane.

  • The external membrane is continuous with the endoplasmic reticulum that is located adjacent to the nucleus.
  • The internal membrane provides nuclear structural support through the presence of a cytoskeleton comprised of fibrous protein.[3][2][4][5][6][7]

Links Image of a Nucleus

MBOC - A cross-sectional view of a typical cell nucleus


Function

The nucleus has many and varied functions such as to monitor and coordinate activities undertaken throughout a cell, this is why it is often referred to as the control centre of the cell. Its tasks include initiation of gene expression by the modification of messenger ribonucleic acid (mRNA) and selective gating of specific proteins into the nuclear compartment.[8][9][10]

History

Image of membrane development[3]

Discovery of the nucleus

The nucleus of eukaryotic cells was always going to be discovered; given the advancement of microscopy.. . . note about - telescopes that allow the mysteries to be uncovered.. As the nucleus is such prominent feature in a cell it may have been cited earlier, but it was first described by Austrian botanist Franz Bauer.[11] In 1831 Scottish bottanist Robert Browne popularised its discovery.

History of nuclear membrane development

Prokaryotic cells have the presence of deoxyribonucleic acid (DNA) floating within the cytoplasm throughout its history and today. The development of nucleus involved the invagination of the plasma membrane from prehistoric prokaryotic cells. Evolution has caused the infolding of the plasma membrane causing extracellular fluid (ECF) and plasma membrane constituents to surround DNA. Subsequently, the region of plasma membrane introversion became separate from the external environment creating a partition. The separated membrane structure gradually collapsed and fused together creating a bi-layered lipid membrane that is called the ‘nuclear envelope’ today. Collectively, the total membranous parts of a cell is aggregately called the ‘endomembranous system’.[12]

Nuclear Envelope

Architecture of the Nuclear Envelope[3]

The nuclear envelope is a distinct structure that maintains a unique biochemical compartment away from the cytoplasm. The nuclear envelope controls the movement of molecules through the use of selective channels and pathways. It is possible as amongst the nuclear membrane, there are structures located intermittently with sole purpose of controlling molecule movement through the membrane called ’nuclear pore complexes’. The nuclear envelope is comprised of two constituents namely, the inner membrane and outer membrane. These two membranes are structured similarly and may have an intervening space called the ’perinuclear space’.[1][2][6][7]


  • The outer membrane is comprised of a single layer of phospholipid coat with proteins, cholesterol, lipids and possibility of ribosomes dispersed amongst its fluid structure. The outer membrane may separate from the nuclear membrane and extend into the cytoplasm to form the ‘endoplasmic reticulum’. Due to this phenomenon, it causes the peri-nuclear space to become continuous with the convoluted tubules of the endoplasmic reticulum.[1][2][6][7]


  • The inner membrane is constructed with a variety of different proteins along with one layer of the bilayered phospholipid coat. It has a nuclear cytoskeleton attached at irregular intervals designed to maintain the structure of the nucleus.[3][1][2][6][7]


  • The nuclear lamina is an additional opaque layer located underlying the inner face of the inner membrane. It is relative thin at approximately 20µm composed of proteins called ‘lamins’. The nuclear lamina provides a molecular interface interlinking the chromatin and the nuclear envelope to provide a framework for structural support and mechanical stability.[3][13][6][9]

Lamins are polypeptides belonging to the Type-V Intermediate Filament protein family. Lamins are found in a rod-shaped form within a main coiled region associated with a nitrogen and carbon terminal domains. Mammalian cells comprise of lamins that are encoded according to genes: LMNA, LMNB1 and LMNB2.

  • The LMNA gene allows encoding and the production of lamins A and C.
  • The LMNB1 and LMNB2 genes trigger protein formation of lamins B1 and B2 respectively.

The composition of the nuclear lamina involves mainly nuclear lamins and a large number of lamin variants. The majority of the lamin species within the nuclear lamina is constituted by lamin B1. The nuclear lamina acts as an intermediate linkage between the chromatin and the nuclear envelope of a cell. Consequently, it permits interactions between proteins of nucleosomes within chromatin and the nuclear envelope.[10][7][14][15]

Click here for more information about Lamin B1


There is the presence of various actin-binding proteins (ABP) found within the nuclear envelope designed to regulate actin filament arrangement and structure. A specific actin-binding protein named Spectrin is primarily involved in maintaining nuclear integrity at the region of the nuclear envelope. In addition, it has multiple accessory roles involved in chromatin remodeling, RNA processing, transcription and nuclear export.

Click here for more information about Spectrin.


Links Image of Nuclear Envelope

Nature Medicine - Image - The nuclear membranes include the interconnected but distinct inner and outer nuclear membranes and the nuclear pore membrane

Nuclear Pore Complexes

Detailed view of Nuclear Pore within the Nuclear Envelope[3]

Nuclear pore complexes are dispersed randomly across the nuclear envelope creating a passageway through the nuclear envelope connecting the cytoplasm to the nucleoplasm. It is a fluid-filled chamber maintaining the essential physiological task in cell survival. The size of a nuclear pore may extend up to 120nm, which is 30 times greater than a ribosome. Nuclear pore complexes are built on a central framework that is eight-fold symmetrical within the nuclear envelope called a ‘spoke complex’. It ensures that the entire structure is embedded in the nuclear envelope. The cytoplasmic and nuclear sides of the ‘spoke complex’ have subunit components attached called the ‘cytoplasmic ring’ and ‘nuclear ring’ respectively.[3][12][2][16][17]


The aggregate structure of the nuclear pore complex involves the spoke complex along with the cytoplasmic and nuclear rings.

  • The ‘cytoplasmic ring’ has eight cytoplasmic filaments attached to its outermost regions extending into the cytoplasm.
  • The ‘nuclear ring’ appears in an arrangement over the nuclear pore extending into nucleoplasm distally. It has additional interconnected filaments creating a ’nuclear basket’ structure similar to a basketball net.[16][18][19]


The nuclear pore complex has a large central channel and multiple peripheral channels. The central channel permits molecules of up to 26nm to pass through the pore, if it is associated with a nuclear import or export signal (otherwise called ‘nuclear localization signal’). Thus despite the expansive size of the central channel, ribosomes and organelle constituents cannot pass through into the nucleoplasm. The additional peripheral channels enables free bidirectional exchange of small molecules with a size less than 10 nm.[3][20][19][21]


The movement of proteins through the nuclear pore complex, into the nucleoplasm, requires binding of a unique cytosolic protein as an import signal called a ‘nuclear transport receptor’. The protein-receptor complex will need to pass through the diaphragm structure within the central channel of the nuclear pore. The diaphragm is designed to maintain a concentration gradient of the protein by implementing facilitated transport. Subsequent to entry into the nucleoplasm, the nuclear transport receptor detaches from the protein and returns to the cytoplasm by flowing back through the same nuclear pore complex.[22][12][19][21]


Alongside the foundation of the nuclear pore complex, there are filamentous structures that extend outwards into the cytoplasm and nucleoplasm that are ‘cytoplasmic filaments’ and ‘long filaments’.

  • The ‘cytoplasmic filaments’ adhere to the cytoplasmic ring and act as docking sites for proteins to be transferred into the nucleus.
  • The ‘long filaments’ are located on the furthest point of the nuclear ring in groups of eight with lengths up to 100nm. Each long filament is interconnected at their distal ends creating a circle structure called a ’nuclear basket’. This particular region is subject to docking of translocating molecules and permits expansion to accommodate for molecules of greater sizes.[3][12][1][18][20]


The composition of the nuclear pore complex involves 50-100 varieties of protein subunits codenamed ‘nucleoporins’. Nucleoporins are accompanied by ‘integral membrane proteins’ as side attachments anchoring the nuclear pore complexes into the nuclear envelope. The remainder of nuclear pore constituents are soluble proteins with a repetitive sequence motif ‘GLFG’ and/or ‘XFXFG’. In this instance, ‘G’ stands for glycine, ‘F’ for phenylalanine, ‘L’ for leucine and ‘X’ for small polar molecule. These additional proteins have an unknown purpose although are suspected to aid import and export receptors for nucleocytoplasmic transport.[3][23][19][21]


A specific nucleoporin comprising the majority of the Nuclear Pore Complex is Nup 358/RanBP2. It is a large molecular weight protein of 358 kD, with a rod-like appearance, located on the cytoplasmic side of the Nuclear Pore Complex. The Nup 358/RanBP2 protein undertakes multiple cellular functions with roles dependent upon its activity in particular stages of cycle cell.

Click here for more information about Nup 358/RanBP2.


Links Image of Nuclear Pore Complex

Nature Medicine - Image - The nuclear membranes include the interconnected but distinct inner and outer nuclear membranes and the nuclear pore membrane

Nuclear Transport

Nucleus is a distinct structure separated from the rest of the cytoplasmic content by nuclear envelope (NE). It controls the contents inside the nucleus that are important for regulating activity of nuclear proteins of modern eukaryotes. For instance, the transcription factor and protein kinase control the cell in response to the changes from external environment. Transcription factors and protein kinases are synthesised in cytoplasm although they can only be activated within the nucleus. Therefore, the transport of specific proteins into the nucleus is a vital method of controlling cellular gene expression. Other processes such as basal replication, regulating the cell cycle, and circadian rhythms are all depending on the nucleocytoplasmic traffic. [24] [25] [26] [27] [28]

The nuclear pore complex (NPC) is the sole site of material movement across NE during interphase. The NPC controls movement of proteins and RNA–protein complexes by passive and selective transport between the nucleus and cytoplasm. Small metabolites, ions, and globular proteins with sizes up to ~60 kDA are allowed to pass through the water-filled channel in NPCs by diffusion. On the contrary, molecules of larger sizes require selective transportation into the nucleoplasm by binding to a specific targeting signal or nuclear localisation signal (NLS). This type of active transport also supports movement of small RNAs such as tRNA and small proteins like histones. [24][25][27][28]


There are two types of protein transport processes evident during nucleocytoplasmic exchange being nuclear import and export. The majority of proteins are synthesised in the cytoplasm, therefore nuclear import is a dominant form of protein transportation into the nucleus. Moreover, nuclear export is equally crucial in nucleocytoplasmic transport during later parts of protein processing to return products into the cytoplasm such as tRNA, rRNA and mRNA. In addition, some proteins may be required to move across the NE several times to be fully developed into a functional subunit. In comparison to the nucleus, after importing proteins into the rough endoplasmic reticulum (rER), they cannot exit due to the absence of specific signals to initiate transportation back across the plasma membrane to the cytoplasm. Nucleus therefore has a unique means of transportation, having equally important import and export process.

Most protein carriers involving NPCs are the members karyopherins family. This includes importin for importing protein into the nucleus, and exportins involve in export mechanism. This family typically able to bind to nucleoporins and form a complex with the GTP-bound state of the Ran GTPase. So far there are 10 other members of this family that act as importins.

The NLS has the primary role as a selector of specific cargo proteins that may cross NE at any one time. The first NLS to be indentified was in nucleoplasmin associated with a simian virus (SV40) large-T antigen. This classical mono or bipartite NLSs consist of a specific sequence that can recognise and bind import and export receptors and allows the development of the shuttle process occurring between the nucleus and the cytoplasm.


Nonetheless many other nuclear proteins are transported by other mechanisms that often do not invole NLS. One example is hnRNPA1, a protein that can shuttle between the nucleus and cytoplasm and use M9 as its sequence. M9 is recognised by transportin (also known as karyopherinβ2) and does not bind to importinα. However for this page, only classical and well understood NLS pathway will be discussed thoroughly.


Nuclear import

Several nuclear transport factors have been discovered to be involved in nuclear import that include Ran, importin α (also known as karyopherinα, Kapα, and PTAC58),importin β (also known as karyopherin β, p97, and PTAC97), NTF2 (also known as p10 or pp15), and a small GTPase (also known as TC4). It suggests that two Ran molecules are exported per transport cycle.[24][25][27][28]

Steps involve in NLS-dependent import cycle in the nucleus :

  1. The NLS-dependent import substrate binds at the importin-α subunit binding site in the importin α/β heterodimer. Importin α does not have a direct interact with NPC instead being an adapter and bind to importin β which has the function as the carrier of the cargo protein complex. Subsequently, the Importin-RanGDP complex binds cargo proteins containing the specific NLS acting as an initiator of this process.
  2. Cargo protein-receptor trimeric complex binds to the cytoplasmic filament part of NPC via importin β binding domain (IBB domain).
  3. The NLS/importin α/β complex is ready to be transferred into the nucleus where there are high RanGTP concentrations available. The exact translocation mechanism is not yet known. Nevertheless it is known to be an energy-dependent mechanism.
  4. To acquire energy, GDP is exchanged with the free RanGTP that directly binds to importin β. Conformational change occurs and causes dissociation of the trimeric NLS/importin α/β complex. NLS/importin α complex is known as an inert cargo protein, which requires importin β, to be imported across the NE. The heterodimer subunits of importins enter the nucleus together but return to cytoplasm by separate pathways. They are known to be transferred back at very different rates. The dissociation occurs due to the free RanGTP present in high concentrations within the nucleus.
  5. After being exported back to the cytoplasm, RanGTP/importin β meets GTPase activating protein (RanGAP1). This then induces the conversion of RanGTP to GDPbound form and as the result the RanGTP concentration in the cytoplasm decreases. Another cytoplasmic factor, Ran-binding protein (RanBP1) also facilitates this reaction. These steps create the differences of RanGTP concentration’s gradient between NE that allows importin heterodimer to form in the cytoplasm and dissociate in the nucleus.
  6. NLS cargo protein is displaced from importin α and released into the nucleus.
  7. CAS, a specific nuclear export receptor, is required for the transfer of importin α. Importin α forms trimeric RanGTP/CAS/importin α complex.
  8. CAS/RanGTP binds and induces a direct export of importin α into the cytoplasm. This action works independently without the need for an importin β molecule since CAS interacts directly with NPC.
  9. In the cytoplasm, RanGTP dissociates from RanGTP/CAS/importin α complex and becomes hydrolysed into RanGDP. This is an irreversible process triggered by RanGAP1.
  10. The Ran-free CAS now has low affinity for importin α binding and undertakes cleavage. The importin heterodimer left remain associated with importin β is ready for the next cycle of nuclear import.

Nuclear export

The first export carrier to be found was Crm1 (exportin1, or Xpo1 in budding yeast) in human immunodeficiency virus (HIV) protein. Until now, the exact sturcture of exportin has not been known yet. Some of the exportins have been identified such as Crm1/Xpo1 (responsible for few cargo protiens), Tap-1/Mex67 (needed for mRNA export), exportin4, Msn5, exportin-t and so on.

Nuclear export is mainly needed for the shuttle of synthesised RNAs from the nucleus to the cytoplasm. However during the NLS-import mechanism, some subunits may require exportation in opposite direction from the nucleus, such as units of importin α and β. These subunits undertake separate routes allowing alternative methods of import cycles (steps 4 - 10 from Nuclear Import above).

A major export transport from nucleus is the shuttle of tRNA by a CAS-like exportin, exportin-t. It is a special exportin for tRNA undergoing direct binding to RNA, that is regulated by RanGTP. In the nucleus, tRNAs are initially synthesised by RNA polymerase III. After several steps of maturation, tRNA is now ready for export to cytoplasm. Exportin-t binds only to the mature tRNA molecules. This specificity allows 'quality control' of tRNA molecules, avoiding complications within the process of nuclear export.[24][25][27][28]

Nucleolus

Image of nucleolus

The nucleolus is one of the most prominent structures in the nucleus. Its size may vary from 1µm in diameter to up to 10µm depending on the type of organism. In various cases, depending on the reproductive stage of the cell, there may be more than one nucleoli present.[29][30][31]


Under light microscopy, the nucleolus can be seen as dense bodies. It has a variety of nucleic acid stains that stain very differently to the rest of the nucleus. In higher eukaryotes, three main components of the nucleolus can be identified under the electron microscope. These include the dense fibrillar component (DFC), granular component (GC) and fibrillar center material (FC).[29][32][33]


The nucleolus is the major site of ribosomal RNA (rRNA) transcription by RNA polymerase I. These genes encode three of the four RNA species of ribosomes (18S, 5.8S, 28S). The rRNAs are synthesized, processed and assembled with ribosomal proteins to form ribosomes. This cycle occurs and repeats during late telophase, through interphase but breaks down and dissembles when the cell proceeds to the mitosis stage. The nucleolus is also responsible for the synthesis of ribosomal precursors.[29][34][35]


Image of Nucleolus

Chromatin and Chromosome DNA Packing

Chromatin

Chromatin is the segment of the nucleus that encloses all the DNA information of the nucleus within the cell. It coils and condenses to form the chromosomes during cell division. Under light microscopy, chromatin can be seen as a diffused, colored mass during interphase. Yet chromosomes can be clearly distinguished under light microscopy. In eukaryotic cells, chromatin is found inside the nuclei and in prokaryotic cells, it tends to be found within the nucleiod. The main function of chromatin is to package 2m of DNA within a nucleus that is roughly 10µm in diameter. This allows smooth separation and more efficient DNA replication during cell division.[36][37][38][39]


Chromatin is mainly comprised of equal masses of histones proteins and DNA. As shown in electron micrographs, unfolded chromatin is displayed as DNA and histone molecules forming “beads on a string” structure. The binding of each “bead” to its adjacent head form a nucleosome. Nucleosomes are the fundamental repeating units of chromatin, which is a complex including the histone octamer and the 147 bp of DNA. The initial level of chromatin organization has 147 base pairs of DNA wrapped one and three quarters turn around a core histone octamer with two molecules of the four types of core histones H2A, H2B, H3 and H4.[36][37][38][39]


Histones

Histones are the main protein constituents of eukaryotic chromosomes. They are rich in basic amino acids and are highly conserved in evolution. There are four classes of histones based on content of basic amino acids:


H1 – very lysine rich

H2A & H2B – moderately lysine rich

H3 & H4 – arginine rich


All five histones have a globular domain and extended flexible portions. The core histones (H2A, H2B, H3, and H4) have structured domains that are composed of histone fold motif and adjacent alpha-helix structures. They also have a very basic N-terminal domain; H2A, H2B and H3, in particular, have a shorter C-terminal domain. These core histones are replication-dependant where its expression is regulated in the cell cycle, whereas the linker histone, H1, has genes encoding minor histone variants and expression only occurs at basal level. There are two ways to distinguish core histones from linker histones and other genes transcribed by RNA polyermase II. Firstly, replication-dependant histone genes (core histones) have an absence of introns. Secondly, the mRNAs produced do not have poly(A) sequences and they end with highly conserved stem loop structure.[40][41]


Overall Structure of Core Histones

Assembly of Histone Octamer

DNA Packaging

During mitosis, a parent cell divides into two daughter cells that are genetically identical. Any difference will lie outside their DNA; this means the differences interpreted between the two daughter cells will depend on the environment where the genes are translated. The definition of a gene’s environment is the way it is packaged into the chromatin. This is also when the “beaded string” structure undergoes higher-order packing.


Another molecule of histone, H1, is attached near the head of the bead. With the help of this histone, the DNA wraps twice around the octamer. The nucleosomal repeat is now approximately 200 nucleotides pairs in length. The string of nucleosome coils and forms a chromatin fiber that is around 30nm in diameter. This 30-nm-fiber loops to form the looped domains, which are then attached to a scaffold of non-histone proteins. The last stage is the formation of metaphase chromosomes, where chromatin folds even further, resulting in the high compact chromosomes at metaphase. As the steps of chromatin packing are strictly specific, genes always result in the same location in metaphase chromosomes.


Stages of DNA Packaging

Chromosome

Chromosomes are discovered in a pairs with half derived from the paternal parent and the other half from the maternal parent. Each constituent of a chromosome pair is individually called a homolog. A chromosome is a linear DNA molecule filled with proteins that is found folded repeated.[42][43][12]


Somatic cells have the tendency to contain diploid (2n) chromosomes that are created through the fusion of gametes from haploid (n) chromosomes. Human cells are comprised of 23 pairs of chromosomes that are 22 pairs of autosomes and 1 pair of sex chromosome. Chromosomes contain two arms within each strand; a shorter ‘p’ arm and a longer ‘q’ arm. The human genome contains about 3.4 × 10^9 base pairs (bp) with a total of 30 000 genes condensed into chromosomes. Chromosomes are viewable mainly during mitosis due to packing and unpacking with a length ranging from 3-7µm.[12][42][44]


Region of Chromosomal Territories within the Nucleus[3]

Chromosomes during interphase are maintained into compartments within the nucleus called ‘chromosomal territories’. Different chromosomes are positioned in accordance to their relevant level of gene content and stage within the chromatin replication process. Chromosomes that are considered gene-poor and activate in late stages of chromatin replication are preferentially found closer to nuclear envelope. In opposition, gene-rich chromosomes required for early stages of chromatin replication are located closer to centre of nucleus.[12][42][45][46]


Image of Chromosomal Territories

The Cell - Chromosome Territories


The pair of sex chromosomes located in the DNA is the main determinant of gender. Females inherit one X chromosomes from the mother and another X chromosome from the father. On the other hand, males inherit one X chromosome from the mother and a Y chromosome derived from father.[12][46]


Chromosomes are structured in a complex fashion with a composition of the following subunits:

  • heterochromatin
  • euchromatin
  • centrosomes
  • telomeres


Image of a Chromosome


Banding of chromosomes

Giemsa staining is a revolutionary banding technique that allows individual chromosomes to be viewed through exposure of chromosomes to trypsin. It produces two distinct colours either Giemsa dark (G) or Giemsa light (R) bands that discriminates against higher order structures in chromosomes required for replication during transcription.[42][47]

  • The light G bands (R) comprise of a high degree of guanine and cytosine with gene-rich concentrations for maintenance and housekeeping. Alongside this gene composition, short intersperse repetitive DNA sequences (SINE) repeats are also unique to these regions.
  • The dark G bands consists of a high proportion of adenine and thymine that are gene-poor although selectively hold tissue-specific genes. The foundation of these regions involves long interspersed repetitive DNA sequence (LINE) repeats.

G and R bands are found localised in different domains with G bands tending to be located nearer to the nuclear envelope and R bands being centralised in nucleus.[42][47]


Deoxyribonucleic Acid (DNA)

Karyotype of Chromosomes[3]

Deoxyribonucleic acid is the functional unit of carrying genetic information of an organism. The physical structure has two complementary chains of nucleotides arranged in a spiral anti-parallel fashion called ‘DNA strands’. The union of the strands is maintained by hydrogen bonds located regularly on the complimentary chains. The deoxyribonucleic acid (DNA) chains extends to a length of 2 metres long and is 10µm in diameter with multiple convolutions, loops and coils through proteins. DNA composition involves 23 pairs of ‘nuclear chromosomes’ numbered from largest to smallest when lined up in a karyotype.[12][42][46]


Each nucleotides have a 5-carbon sugar called a ‘ribose’ connected to a phosphate group and a nitrogen base.

  • The nitrogen bases are called nucleotides, which may be adenine (A), cytosine (C), guanine (G) or thymine (T). The nitrogen base acts as a substituent group attached to the sugar backbone of a DNA strand.

The DNA backbone involves the ribose and phosphate groups alternating in a polypeptide fashion i.e. –(sugar-phosphate-sugar-phosphate)n–.[12][44][46]


The sugar-phosphate backbones are located at the periphery with the nitrogen bases acting as intermediates between the two strands joined by hydrogen bonds. It allows a ladder-like structure to be formed with each rung comprised of two complimentary nucleotides called ‘base pairs’. It is designed in this particular manner to allow a regular structure with nucleotides being equidistant along the entire double helix.[43][44]


Image of the Sugar Phosphate Backbone


The DNA double helix interweaves with one full helical turn constructed every ten bases in an anti-parallel complementation of DNA strands oriented opposite each other. Each nucleotide may be classified as either a purines or pyrimidines that are double ring bases and single ring bases respectively. In DNA, adenine and guanine are purine bases whilst cytosine and thymine are pyrimidine bases. Base pairs are formed with two nucleotides bound together with a purine nucleotide and a pyrimidine nucleotide. An equidistant between DNA strands is achieved through the pairing of thymine with adenine and guanine with cytosine.[12][44][46]


Image of DNA


The degree of DNA being twisted is called ‘DNA supercoiling’ dependent upon the activity of enzymes called ‘topoisomerases’. These enzymes attempt to promote ‘negative supercoiling’ causing the DNA double helix to relax and become less twisted. Alternatively, inactivity increases twisting of DNA called ‘positive supercoiling’ bringing each base pair of the DNA double helix closer together.[48][49]

Deoxyribonucleic acid can be divided into a set of linear chromosomes called a ‘genome.’ A human genome is composed of the genetic material of an entire cell, which contains 3 billion base pairs configured into 46 chromosomes. Heredity information is coded onto DNA strands called ‘genes’ that can potentially produce a biological message through transcription and translation.[42][43]




Heterochromatin and Euchromatin

Chromosomes are not evident during an interphase nucleus although it may be found as specific parts of chromatin that can be detected by staining. The staining of the fragments allows identification of ‘heterochromatin’ and ‘euchromatin’.

  • Heterochromatin involves chromosomal pieces that can be stained and traced throughout a cell cycle.
  • Euchromatin differs to heterochromatin as it becomes invisible during telophase and interphase.

Staining allows heterochromatin to be differentiated from euchromatin as it has few activated genes and tends to contain primarily deoxyribonucleic acid (DNA) sequences.[42][45]




Centromere

Region of a centromere[3]

Centromeres are chromosomal substructures that maintain the segregation of chromosomes during meiosis and mitosis. Centromeres are found singularly on the majority of eukaryotic chromosomes attaching sister chromatids. It is involved in key cell cycle processes including spindle microtubule attachment, mitotic checkpoint control, sister-chromatid cohesion and cytokinesis. Transmission of the centrosome is evident at an identical chromosomal location, before and after cell division. Control of centromere structure and function is regulated through the presence of centromeric deoxyribonucleic acid (DNA) sequences rather than primary DNA sequences. The composition of a centromere involves tandem repeated satellite DNA sequences found similarly in organisms.[50][51][52]


Location of Centromere


In human chromosomal centromeres, α-satellite DNA is the main constituent; made of 169-172 base pair monomers and specific higher-order repeat units. Each higher-order repeat may appear in variations of up to 30 diverged monomers in length and is influenced primarily by intrachromosomal processes. The aggregate structure of the α-satellite DNA involves several thousand multimer tandem repetitions. The α-satellite DNA in humans can be separated into different categories of suprachromosomal families, each with a unique set of monomeric types. Each subfamily is associated with specific regions having alternate compositions that are located similarly on the chromosome.[51][52][53]


Image of Centromeres

MBOC - Centromere


Centromere-associated Proteins

The majority of centromere-associated proteins remain highly conserved and contribute towards centromere structure and function. These centromere proteins (CNEPs) may be identified as either CENP-A, CENP-B, CENP-C, CENP-D, and CENP-E and remain part of the centromere in the cell cycle.[51][54][53]

  • CENP-A is a histone H3 that may be found at the kinetochore plate within centromeres and binds α-satellite DNA. It is arranged on the surface of centromeric heterochromatin designated for centromere construction and function.
  • CENP-B plays an essential role in achieving stability in centromere development and structure.
  • CENP-E is a kinesin-like motor protein located at the kinetochore plate that belongs to a second class of centromeric proteins.

Sister chromatids are joined at centromeres by cohesion by many proteins. These proteins vary along the chromosomal arms and centromeres and activated/deactivated throughout the cell replication process.[51][54][53]




Telomeres

Localisation of Telomere on Chromosomes[3]

Telomeres are specialized DNA-protein complexes located on the ends or ‘caps’ of human chromosomes. The function of telomeres involves constantly building and breaking down of terminal DNA and prevention of chromosomal degradation. A unique ribonucleoprotein enzyme called ‘telomerase’ is necessary to regulate long term telomeric DNA production and repair. Telomeres are comprised of dynamic DNA tandem repeats of 6 base pairs, which contain clusters of guanine (G) residences on chromosomal termini. It is evident through the presence of TTAGGG in humans. Telomeric DNA-binding proteins may also attach to telomeric DNA to signal production of high-order DNA.[55][56][57]


The terminal region on the linear DNA molecule involves telomeric DNA that replicates by telomerase and traditional semiconservative DNA replication enzyme machinery. Telomerase is a vital ribonucleoprotein enzyme when activated attaches telomeric DNA onto pre-existing telomere DNA of chromosomes. It allows the protection of chromosomal material by ensuring that chromosomal DNA is replicated properly. In addition, telomerase repairs broken DNA ends by adding telomeric repeats onto damaged areas. Dysfunctional telomerase triggers telomere shortening and destroys the ability for cellular proliferation.[56][57][58]


Image of Telomeres on a Chromosome


Telomerase is constituted by two components: ‘human telomere reverse transcriptase (hTERT)’ acting as a catalyst and ‘human telomerase ribonucleic acid (hTR)’ as a RNA part. Telomerase implements the RNA component act as a template for reverse transcription that builds DNA tandem (TTAGGG) repeats onto chromosomal telomeres. It helps maintain and regulate telomere length thus in its absence, somatic cells may lose telomeric sequences naturally by cell division. Cellular ageing has been discovered to cause telomere shortening as telomerase activity decreases and continued loss of telomeric sequences occurs.[56][57]


In eukaryotic human cells, telomeric DNA comprises of up to 20 kilobases of DNA tandemly repeated (TTAGGG) sequences. It involves thymine, adenine and guanine. It may be found alongside two proteins being: telomeric repeat binding factor 1 (TRBF1) and 2 (TRBF2). Coordinated expression of these proteins act upon telomeric DNA directly, subsequently, controlling telomere length.[56][57]


Normal somatic cell division cause telomeres to shorten by 50-200 bp due to inability for DNA to replicate particular parts at each end. This event is called the ‘end replication problem’ due to progressive loss of telomeric sequences.


Telomeric regions experience increased genetic recombination leading to varying gene exchanges and imbalances of gene dosage. Recombination during meiosis requires telomeres to regulate chromosome pairing by acting as the primary region of chromosomal union and synapse. Therefore, it is important for controlling homologous chromosome pairing. Chromosomal pairing is displayed through telomere movement towards the nuclear envelope clustering in a ‘bouquet formation’. This permits an exchange of material between non-homologous chromosomes.[56][57][58]


Subtelomeric regions

Subtelomeric regions are located proximal to the human DNA tandem repeated sequences (of TTAGGG). These may also be known as telomere-associated DNA with segments of shared homology between different chromosomes. Subtelomeric regions are generally a combination of shared repeated DNA alongside additional sequence homologies. It has the main purpose of sequencing information from various telomere regions.[56][57][58][59]


There are two subdomains in telomeres that being a ‘distal subdomain’ and ‘proximal subdomain’.

  • The distal subdomain is located nearer to the TTAGGG repeats (located distally). It involves many short sequences with shared homologies shorter than 2 kilobases.
  • The proximal subdomain differs with longer segments of up to 40 kilobases of shared homologies present in chromosomes.

The proximal and distal subdomains are intervened by many degenerate TTAGGG repeats and replication-specific sequences that helps arrange telomere repeats.[56][57][58][59]


Unique Sequence DNA

Unique sequence DNA is specifically positioned adjacent to subdomain telomeric regions located further away from the end of the chromosome. The length of unique sequence DNA ranges up to 300 kilobases and usually is the region of highest density of genes.[60]

Eukaryote Gene Expression

DNA replication

DNA in eukaryotes is formed from two strands of nucleotides, joining to give rise to the DNA double helix. DNA replication is when an entire double stranded DNA is duplicated. A new DNA double strand helix is formed, and is passed on to the newly divided daughter cells along with the existing parental strand. This process is compulsory for cell reproduction prior to cell division. The steps of DNA replication are summarized as follows:

1. Separation of DNA double helix – The hydrogen bonds between the bases of the double strand helix are to be unwounded. Normally this takes place at between the Adenine and Thymine bond as they are only linked by two hydrogen bonds, whereas there are three between Cytosine and Guanine. The corresponding enzyme for this reaction is helicase. The splitting occurs at the “origin” and the separated structure is now called the “replication fork”. Once separated, the two DNA strands act as a template for the development of the new strand.

2. Binding of RNA Primase – The RNA primase binds to the 3’-5’ parent chain. The function of RNA primase is to recruit RNA nucleotides (precursors for binding of DNA nucleotides), which bind to DNA nucleotides of the 3’-5’ strand. This occurs because of the hydrogen bond attraction between the bases.

3. Elongation – This procedure is different for the 3’-5’ and 3’-5’ chains.

4. Elimination of RNA primers – RNA primers are removed by DNA PoII – exonuclease. DNA polymerase then closes the gaps by the addition of complementary nucleotides. The phosphate sugar backbone gap is closed by the addition of phosphate by DNA ligase.

5. Termination -

Variations of Nucleus in Different Cells

Some cells specialised cells such as red blood cells (RBCs) do not contain a nucleus. O

Nuclear suborganelles / subcompartments / subdomains

Nuclear bodies (NBs) are the distinct suborganelles present within higher eukaryotes nucleus, particularly during the interphase. These substructures are surrounded mostly by the chromosomes yet consisting specific protein and RNA factors associated within them. They do not have membrane seperating the contents from nucleoplasm, unlike cytoplasmic compartments (mitochondria, lysosomes and so on) in cytoplasm. They react to specific cellular signals between other NBs and involve in nuclear gene expression. Alteration within their constitution can result in various disease phenotypes. NBs involve in the role of sythesising, processing, modificating or transporting various cellular RNAs. With the size of 0.2–1.0 μm in diameter, mammalians usually contain 10-30 NBs in their nuclei.[61][62]


Classification of Nuclear Bodies

Nuclear Bodies can be classified into different types based on their morphological structure and constituents that may be either Cajal Bodies, PML Bodies or Speckle/IGC.

Cajal Bodies

Cajal Bodies (also known as nucleolar accessory bodies or coiled body (CBs)) are typically found in the nucleoplasm of animals and plants cells. It was first found to be the accessory bodies of the nucleolus in 1903. Coiled bodies (CBs) as what the name explains, appear in the form of tangled coiled electrodensed threads under electron microscope, with the size of ~0.5 μm in diametre. The complex structure of CBs keep changing depending on the phases: disassembling throughout mitosis, reassembling during mid G1 after nucleologenesis and at the beginning of transcription process. It is also known to consist of high snRNPs concentration.

Cajal Bodies contain:

- three major classes of intranuclear small nuclear ribonucleoproteins (snRNPs) such as :

  1. Spliceosomal U1, U2, U4, U5 and U6 snRNPs -- for pre-mRNA splicing
  2. U7 -- for 3' maturation of histone mRNA
  3. U3 and U8 small nucleolar RNAs (snoRNAs) -- for pre-tRNA's processing

- epitotes such as p80 coilin (CBs signature's protein) and fibrillarin

A study done by Carvalho et al. (1999) stated that CBs consist of protein components such as the Survival Motor Neuron gene (SMN) and its associated protein, SIP1. Both involve in the biogenesis, trafficking, and recylcing of snRNP, which form a direct link to the function of CBs SMN [63]


The exact functions of Cajal bodies remain elusive due to its widely distributed components within other nuclear bodies and nucleoplasm. However many research had shown that CBs have some function such as :

  • Guiding RNAs in site-specific synthesis of 2’-O-ribose-methylated nucleotides and pseudouridines in the RNA polymerase II-transcribed U1, U2, U4 and U5 spliceosomal in the biogenesis of small nuclear RNAs (snRNAs). It is the mediators of post RNA transcription steps. [61] [64]
  • Containing splicing factor for RNA maturation and cell cycle control proteins.
  • Recruiting processing factors to the sites of histone pre-mRNA transcription

Gems

PML bodies

Promyelocytic leukaemia (PML) nuclear bodies is also known as ND10 (nuclear domain 10) or PODs (PML oncogenic domains). The structure is approximately 0.5 um in diameter. They have various functions because of their diverse protein make-ups, such as, the most prominent ones, retinoblastoma protein Rb, Sp100 and PIC1/SUMO-1. These proteins work together to form the proper NB structure and can act as the transcriptional regulator and nuclear storage depots.

Firstly, PML needs to be SUMOylated by SUMO-1 in order to localise in the nuclear body. After being modified, it can then recruits other components to form the NBs.

PML NBs invovles in the disease of acute promyelocytic leukaemia (APL) and often targeted by viral infections.

PML has the ability to move by a metabolic-energy-dependent mechanism, that is usually used by organelles outside the nucleus.

PML bodies

Speckle/IGC

  • PIKA and PTF domain
  • Paraspeckles



Mutation and Disease

The nucleus plays a major role in a cell and contains a large number of different proteins. As the result, it is very susceptible to mutations in its proteins with a small number triggering progression of disease.


Lamin mutation

One of the major structures of nucleus is nuclear envelope which consists of different layers of membrane. Protein lamins as the constituent of the inner nuclear membrane contain LMNA loci which can cause several inherited disease if mutation occurs.

Specifically Lamins A and C mutation can result in 4 major categories of diseases such as:

1) Striated Muscle Disease

  • Autosomal Dominant Emery-Dreifuss Muscular Distrophy

2) Partial Lipodystrophy Syndromes

  • Dunnigan-type Partial Lipodystrophy

3) Peripheral Neuropathy

  • Charcot-Marie-Tooth Disorder Type 2B1

4) ‘Premature Ageing’ Syndromes

Nuclear Bodies Related Diseases

  • Acute Promyelocytic Leukaemia (APL)

In the patient suffer from APL, the nuclei appear to contain oncogenic fusion between PML protein and retinoic acid receptor alpha (RARα) derived from the t(15,17)chromosomal translocation. PML with other NBs components are delocalised and altering the normal nuclear structure. The expression from PML-RARα results in the disruption of NB structure, that can be restored by the ligand of RARα, retinoic acid (RA). RA works by inducing the degradation of PML-RARα fusion protein and rearrange the NBs components into their normal position. Therefore it is often used as the clinical treatment for the patient that induce complete remission of the disease.[65][66]



Current Nuclear Research

Glossary

  • centromere – main contriction of a chromosome as point of attachment of spindle fibres; important for chromosome movement in mitosis
  • chromatin – genetic material found in the nucleus containing deoxyribonucleoprotein
  • chromosome – genetic material within the cell nucleus involved in mitosis and meiosis
  • chromosomal territories – compartments of the nucleus that specific chromosomes occupy during interphase
  • deoxyribonucleic acid (DNA) – nucleic acid containing deoxyribose as sugar component predominantly found in nuclei and mitochondria of eukaryotic cells and loosely bound to proteins; required for chromosome development
  • double helix - helical structure involving two strands of deoxyribonucleic acid linked by hydrogen bonds
  • endoplasmic reticulum (ER) – network of cytoplasmic tubules or flattened sacs that can have ribosomes attached to its surface
  • enzyme – acts as a catalysts in chemical reactions in other substances without being changed during the process
  • euchromatin - dispersed lightly staining form of chromatin
  • extracellular fluid (ECF) – fluid residing outside of a cell
  • genetic recombination - progeny of combinations of genotypes created through a process of crossing-over
  • genome – complete set of chromosomes derived from a parent
  • Giemsa stain - compound of methylene blue-eosin and methylene blue used for demonstrating chromosome G bands
  • heterochromatin - condensed, readily stainable aggregates of chromatin
  • lamin – polypeptides belonging to Type-V intermediate family; integral in providing structural support for nuclear envelope
  • messenger RNA (mRNA) – RNA reflecting exact nucleoside sequence of active DNA carrying a ‘message’ to cytoplasmic proteins to create specific amino acid sequences
  • nucleus – round or oval mass of protoplasm founding within the cytoplasmic compartment of an animal or plant cell; it is surrounded by a nuclear envelope
  • nuclear envelope – double membrane at boundary of nucleoplasm; has nuclear pore complexes integrated into its structure
  • nuclear lamina – a protein-rich layer underlying the inner layer of the nuclear membrane of interphase cells
  • nuclear pore complex – protein-based structure situated in the nuclear envelope regulating transport between the nucleus and cytoplasm
  • nucleotide – compound created with a nucleic acid, a sugar group and a phosphoric group
  • perinuclear space (cisterna caryothecae) – space between inner and outer membranes of the nuclear envelope
  • telomerase – a reverse transcriptase comprising an RNA template and a catalytic protein component; absent in normal ageing somatic cells
  • telomere – distal region of a chromosome arm


  • Nuclear Localisation Signal (NLS) - specific sequence used during active transport between nucleocytoplasm

NES = Nuclear Export Signal

GTP = guanosine triphosphate

GDP = guanosine diphosphate

PML = promyelocytic leukaemia protein

POD = PML oncogenic domains

snRNP = small nuclear ribonucleoproteins

APL = Acute Promyelocytic Leukaemia

  • importin α - soluble transport factor acting as an adapter protein in the import process that binds directly to an NLS

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2009 Group Projects

--Mark Hill 14:02, 19 March 2009 (EST) Please leave these links to all group projects at the bottom of your project page.

Group 1 Meiosis | Group 2 Cell Death - Apoptosis | Group 3 Cell Division | Group 4 Trk Receptors | Group 5 The Cell Cycle | Group 6 Golgi Apparatus | Group 7 Mitochondria | Group 8 Cell Death - Necrosis | Group 9 Nucleus | Group 10 Cell Shape