3. Chapter 7 - (Kabwe) PDF

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Chapter 7: Organization of DNA on ChromosomesAnd Cell CycleHuman Chromosome Karyotype7 Chromosome RegionsCentromeric DNATelomeric DNAProtect Your DNA As you age, the ends of your chromosomes become shorter. This makes you more likely to get sick. But lifestyle changes can boost an enzyme that makes...

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Chapter 7: Organization of DNA on Chromosomes And Cell Cycle Human Chromosome Karyotype

7.1 Chromosome Regions

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Centromeric DNA

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View of the attachment of the kinetochore

Are Telomeres the Key to Aging and Cancer? 

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Inside the nucleus of a cell, our genes are arranged along twisted, double-stranded molecules of DNA called chromosomes. At the ends of the chromosomes are stretches of DNA called telomeres, which protect our genetic data, make it possible for cells to divide, and hold some secrets to how we age and get cancer. Telomeres have been compared with the plastic tips on shoelaces, because they keep chromosome ends from fraying and sticking to each other, which would destroy or scramble an organism's genetic information. Yet, each time a cell divides, the telomeres get shorter. When they get too short, the cell can no longer divide; it becomes inactive or "senescent" or it dies. This shortening process is associated with aging, cancer, and a higher risk of death. So telomeres also have been compared with a bomb fuse.

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Telomeric DNA

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Protect Your DNA 

As you age, the ends of your chromosomes become shorter. This makes you more likely to get sick. But lifestyle changes can boost an enzyme that makes them longer. Plus, studies show diet and exercise can protect them. The bottom line: healthy habits may slow aging at the cellular level.

7.2 DNA Organization of Eukaryotic Chromosomes Eukaryotic Chromosomal Organization  

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Many eukaryotes are diploid (2N). The amount of DNA that eukaryotes have varies; the amount of DNA is not necessarily related to the complexity (Amoeba proteus has a larger amount of DNA than Homo sapiens). Eukaryotic chromosomes are integrated with proteins that help it fold (protein + DNA = chromatin). Chromosomes become visible during cell division. DNA of a human cell is 2.3 m (7.5 ft) in length if placed end to end while the nucleus is a few micrometers; packaging/folding of DNA is necessary. 2 main groups of proteins involved in folding/packaging eukaryotic chromosomes. Histones = positively charged proteins filled with amino acids lysine and arginine that bond. Non-histones = less positive. Histone Proteins o Abundant. o Histone protein sequence is highly conserved among eukaryotes—conserved function. o Provide the first level of packaging for the chromosome; compact the chromosome by a factor of approximately 7. o DNA is wound around histone proteins to produce nucleosomes; stretch of unwound DNA between each nucleosome. Non-Histone Proteins o Other proteins that are associated with the chromosomes. o Many different types in a cell; highly variable in cell types, organisms, and at different times in the same cell type. o Amount of non-histone protein varies. o May have role in compaction or be involved in other functions requiring interaction with the DNA. o Many are acidic and negatively charged; bind to the histones; binding may be transient.

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Model of Chromatin Structure     

Chromatin is linked together every 200 bps (nuclease digestion). Chromatin arranged like “beads on a string” (electron microscope). 8 histones in each nucleosome. 147 bps per nucleosome core particle with 53 bps for linker DNA (H1). Left-handed superhelix.

Histone Protein

A possible nucleosome structure. Linker DNA

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Nucleosomes are connected together by linker DNA and H1 histone to produce the “beads-on-a-string” extended form of chromatin. 11nm chromatin is produced in the first level of packaging.

Eukaryotic Chromosomal Organization

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Histone Proteins   

DNA is further compacted when the DNA nucleosomes associate with one another to produce 30nm chromatin. Mechanisms of compaction is not understood, but H1 plays a role (if H1 is absent, then chromatin cannot be converted from 10 to 30nm). DNA is condensed to 1/6th its unfolded size.

Packaging of nucleosomes into the 30nm chromatin fiber.      

Compaction continues by forming looped domains from the 30nm chromatin, which seems to compact the DNA to 300nm chromatin. Human chromosomes contain about 2000 looped domains. 30nm chromatin is looped and attached to a non-histone protein scaffolding. DNA in looped domains are attached to the nuclear matrix via DNA sequences called MARs (matrix attachment regions). MARs are known to be near regions of the DNA that are actively expressed. Loops are arranged so that the DNA condensation can be independently controlled for gene expression.

Model for the organization of 30nm chromatin fiber into looped domains that are anchored to a non-histone chromosome scaffold.

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The many different orders of chromatin packing that give rise to the highly condensed metaphase chromosome.

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DNA Compaction  

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Level of DNA compaction changes throughout the cell cycle; most compact during mitosis and least compact during S phase (DNA synthesis). 2 types of chromatin; related to the level of gene expression: 1. Euchromatin—defined originally as areas that stained lightly. More active areas of DNA. 2. Heterochromatin—defined originally as areas that stained darkly. Euchromatin: chromosomes or regions therein that exhibit normal patterns of condensation and relaxation during the cell cycle. o Most areas of chromosomes in active cells. o Usually areas where gene expression is occurring. Heterochromatin: chromosomes or regions therein that are condensed throughout the cell cycle. Provided first clue that parts of eukaryotic chromosomes do not always encode proteins.

DNA Packaging: Nucleosomes and Chromatin

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Article by Anthony T. Ammuziato: http://www.nature.com/scitable/topicpage/dnapackaging-nucleosomes-and-chromatin-310

7.3 Unique-Sequence and Repetitive-Sequence DNA in Eukaryotic Chromosomes Three different types of sequences have been named: Unique (or single-copy, present in one to a few copies per genome), moderately repetitive (present in a few to as many as 103 to 105 times in the genome), and highly repetitive (present as many as 105 to 107 times) although there are really no discrete boundaries between them. In prokaryotes, with the exception of the ribosomal RNA genes, tRNA genes, and a few other sequences, all of the genomes are present as unique-sequence DNA. Eukaryote genomes on the other hand consist of both unique sequence and repetitive – sequence DNA with the latter typically being quite complex in number of types, number of copies, and distribution. The human genome consists of about 64% unique-sequence DNA, 25% moderately repetitive DNA, and 10% highly repetitive DNA. By contrast the green frog has 22% unique sequence DNA, 67% moderately repetitive DNA, and 10% highly repetitive DNA. Unique-Sequence DNA: unique sequences also called single-copy sequences are defined as sequences present as single copies in the genome. Most of the genes that code for proteins in the cell are in the unique-sequence class. Repetitive – Sequence DNA: this group is divided in two sub-groups: tandemly repeated DNA sequences and the dispersed repeated DNA sequences. 1. Tandemly Repeated DNA: refer to sequences that are tandemly arranged (arranged one after the other) in the genome. They are quite common in eukaryotic genomes, and a number of different examples are known. The genes for ribosomal RNA and transfer RNA. Some histone genes except in mammals and birds tend to be linked. Some tandemly repeated genes are related, but not identical. For example, in vertebrates, hemoglobin consists of two copies each of an α-globin and β-globin polypeptide. There is an α-globin family of genes and a β-globin family of genes. In human, the three genes of the α-globin family are located on chromosome 16 and the five genes of the β-globin family are located on chromosome 11. Some tandemly repeated sequences are not associated with genes. In fact, the greatest amounts of tandemly repeated DNA are associated with centromeres and telomeres. At each centromere, for example, hundreds to thousands of copies of simple, relatively short, tandemly repeated sequences (i.e. highly repetitive sequences) may be found. Indeed, a significant proportion of the eukaryotic genomes may be comprised of the highly repeated sequences found at centromeres. 2. Dispersed repeated sequences: Refers to those that are dispersed in the genome, rather than being tandemly arranged. Examples of both gene sequences and non-gene sequences are found in this class of repeated sequences. Typically, dispersed repeated

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genes are present in relatively low copy number, often less than 50. Another type of dispersed repeated sequence genes is the transposon, a DNA element that is capable of moving to other locations in the genomes. Often small numbers of families have very high copy numbers and make up most of the dispersed repeated sequences in the genome. Two general interspersion patterns are encountered. In one, the families have sequences 100 to 500 bp long. These sequences are called SINESs for short interspersed repeats. In the other, the families have sequences about 5,000 bp or more, long. These sequences are called LINESs for Long Interspersed repeats. All eukaryotic organisms have SINESs and LINESs, although the relative proportions vary considerably. A well-studied SINE family is the Alu family of certain primates (most abundant in human males – 1 million copies). This family named for the restriction site for AluI found in the repeated sequences. In human, the Alu family is the most abundant SINE family in the genome, consisting of 300-bp sequences repeated as many times as 9 x 105 times, and comprising about 9% of the total haploid DNA. Alu repeats along with other SINEs and also many LINES are found in a variety of places, including in introns, in the DNA flanking genes, and in satellite DNA. But there is little knowledge about the function (s) of SINEs and LINEs. One hypothesis is that most of these sequences have no function at all. G-Banding

Genetic Map of Human Chromosome 1

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Genetic map of human chromosome 1. At the left is a drawing of the chromosome. The two vertical lines at the right represent genetic maps derived from studies of recombination in males and females. Between the genetic maps are the order and location of 58 loci, some of which are genes (red) and others (blue) that are genetic markers detected using recombinant DNA techniques. The map in females is about 500 cM long, and in the males, it is just over 300 cM. This is a result of differences in the frequency of crossing over in males and females. This map provides a framework for locating genes on the chromosome as part of the Human Genome Project.

7.4 DNA Cell Cycle    

At the G1/S, G2/M, and M check points, cells decide whether to proceed to the next stage of the cell cycle. Regulation of the cell cycle progress is mediated by cyclins and cyclin-dependent kinases (CDKs) that regulate synthesis and destruction of cyclin proteins. If a problem is noted at the beginning of the cell cycle, cyclin can hold the cell in a quiescent state. This allows the DNA to be repaired. If a problem is noted at the end of the cell cycle, cyclin can not hold the cell and will force the cell to undergo apoptosis.

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Cyclin and Cyclin Dependent Kinases

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Mutations in these repair genes will allow more genetic damage to enter into the system. This build up of damage will eventually lead to cancer. All cancer is related to problems in the cell cycle. Mutations in genes that effect the cell cycle will often times lead to increased risks of developing cancer.

Other Readings Tumor Protein p53, also known as p53 

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Cellular tumor antigen p53 is any isoform of a protein encoded by homologous genes in various organisms, such as TP53 (humans) and Trp53 (mice). This homolog is crucial in multicellular organisms, where it prevents cancer formation, thus functions as a tumor suppressor. As such, p53 has been described as “the guardian of the genome” because of its role in conserving stability by preventing genome mutation. Hence TP53 is classified as a tumor suppressor gene.

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Cyclins Progress in the eukaryotic cell cycle is driven by oscillations in the activities of CDKs (CyclinDependent Kinases). CDK activity is controlled by periodic synthesis and degradation of positive regulatory subunits, Cyclins, as well as by fluctuations in levels of negative regulators, by CKIs (CDK Inhibitors), and by reversible phosphorylation. The mammalian cell cycle consists of four discrete phases: (1) S-phase, in which DNA is replicated; (2) M-phase, in which the chromosomes are separated over two new nuclei in the process of mitosis. These two phases are separated by two so called “Gap” phases, G1 and G2, in which the cell prepares for the upcoming events of S and M, respectively (Ref.1). The different Cyclins, specific for the G1-, S-, or M-phases of the cell cycle, accumulate and activate CDKs at the appropriate times during the cell cycle and then are degraded, causing kinase inactivation. Levels of some CKIs, which specifically inhibit certain Cyclin/CDK complexes, also rise and fall at specific times during the cell cycle. A breakdown in the regulation of this cycle leads to uncontrolled growth and contribute to tumor formation. Defects in many of the molecules that regulate the cell cycle also lead to tumor progression. In mammalian cells, different Cyclin-CDK complexes are involved in regulating different cell cycle transitions: Cyclin-D -CDK4/6 for G1 progression, Cyclin-E -CDK2 for the G1-S transition, Cyclin-A -CDK2 for S-phase progression, and Cyclin-A/B-CDC2 for entry into M-phase. Apart from these well-known roles in the cell cycle, several Cyclins and CDKs are involved in processes not directly related to the cell cycle. Cyclins associate with CDKs to regulate their activity and the progression of the cell cycle through specific checkpoints. Disruption of Cyclin action leads to either cell cycle arrest, or to uncontrolled cell cycle proliferation. All Cyclins are degraded by ubiquitin-mediated processes, and the mode by which these systems are connected to the cell-cycle regulatory phosphorylation network, are different for mitotic and G1 Cyclins. The decision by the cell to either remain in G1 or progress into S-phase is the result in part of the balance between Cyclin-E production and proteolytic degradation in the proteosome. Both synthesis and destruction of Cyclins are important for cell cycle progression. The destruction of Cyclin-B by Anaphase-Promoting Complex/cyclosome is essential for metaphaseanaphase transition, and expression of indestructible Cyclin-B traps cells in mitosis. Cyclins-E and A have been implicated in the DNA replication initiation process in mammalian cells.

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7.5 Mutations Causing Loss of Cell-Cycle Control The entry of cells into the cell cycle from a quiescent state and the progression of cells around the cycle are precisely controlled events. This assures that cellular growth and the coordination of DNA synthesis with cell-size increase and cytokinesis are monitored and do not fall out of regulated synchrony. Once a cell progresses past a certain point in late G1, called the restriction point, it becomes irreversibly committed to entering the S phase and replicating its DNA. Cyclins, cyclin-dependent kinases (CDKs), and the Rb protein are all elements of the control system that regulate passage through the restriction point. The ability of these proteins to check cell-cycle progression and hold cells in quiescence (early in cell cycle) or even lead cells to commit suicide (late in the cell cycle) unless conditions are appropriate, means that they can prevent cells from becoming cancerous. Altered regulation of expression of at least one cyclin as well as mutation of several proteins that negatively regulate passage through the restriction point can be oncogenic. Restriction point control. Overexpression or loss of the cell-cycle control proteins commonly occurs in human cancers. Overexpression of Cyclin D1 Gene amplification or a chromosome translocation that places cyclin D1 under control of an inappropriate promoter leads to overexpression of this cyclin in many human cancers, indicating that it can function as an oncoprotein. In certain tumors of the antibody-producing B cells, for instance, the cyclin D1 gene is translocated such that its transcription is under control of an antibody-gene enhancer, causing elevated cyclin D1 production throughout the cell cycle irrespective of extracellular signals (this phenomenon is analogous to the cmyc translocation in Burkitt’s lymphoma cells discussed earlier). That cyclin D1 overexpression can directly cause cancer was shown by studies with a transgenic mouse in which the cyclin D gene was placed under control of an enhancer specific for mammary ductal cells. Initially the ductal cells underwent hyperproliferation, and eventually breast tumors developed in these transgenic mice. Amplification of the cyclin D1 gene and concomitant overexpression of the cyclin D1 protein is common in human breast cancer. Cyclin D is a member of the cyclin protein family that is involved in regulating cell cycle progression. The synthesis of cyclin D is initiated during G1 and drives the G1/S phase transition. Once the cells reach a critical cell size (and if no mating partner is present in yeast) and if growth factors and mitogens (for multicellular organism) or nutrients (for unicellular organism) are present, cells enter the cell cycle. In general, all stages of the cell cycle are chronologically separated in humans and are triggered by cyclin-CDK complexes which are periodically expressed and partially redundant in function. Cyclins are eukaryotic proteins that form holoenzymes with cyclin-dependent protein kinases (CDK), which they activate. The abundance of cyclins is generally regulated by protein synthesis and degradation through an APC/C dependent pathway.

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Cyclin D is one of the major cyclins produced in terms of its functional importance. It interacts with four CDKs: Cdk 2, 4, 5, and 6. In proliferating cells, cyclin D-Cdk4/6 complex accumulation is of great importance for cell cycle progression. Namely, cyclin DCdk4/6 complex partially phosphorylates retinoblastoma tumor suppressor protein (Rb), whose inhibition can induce expression of some genes (for example: cyclin E) important for S phase progression. Role of Cyclin D in Cancer Given that many human cancers happen in response to errors in cell cycle regulation and in growth factor dependent intracellular pathways, involvement of cyclin D in cell cycle control and growth factor signaling makes it a possible oncogene. In normal cells overproduction of cyclin D shortens the duration of G1 phase only and considering the importance of cyclin D in growth factor signaling, defects in its regulation could be responsible for absence of growth regulation in cancer cells. Uncontrolled production of cyclin D affects amounts of cyclin D-Cdk4 complex being formed, which can drive the cell through the G0/S checkpoint, even when the growth factors are not present. Overexpression can happen in one of three ways: (1) as a result of gene amplification, (2) impaired protein degradation, or (3) chromosomal translocation. ...