Chapter 9 Chromosomes Jocelyn E Krebs Figure 09

Chapter 9 Chromosomes Jocelyn E. Krebs

Figure 09. CO: Microphotograph of living cells undergoing mitosis. Individual chromosomes lined up at the metaphase plate or separating are visible in several cells. © Dimarion/Shutter. Stock, Inc.

9. 1 Introduction nucleoid – The structure in a prokaryotic cell that contains the genome. The DNA is bound to proteins and is not enclosed by a membrane. chromatin – The state of nuclear DNA and its associated proteins. • chromosome – A discrete unit of the genome carrying many genes. Each consists of a very long molecule of duplex DNA and an approximately equal mass of proteins. – It is visible as a morphological entity only during cell division. – Tight packing (5 -10 fold than chromatin) Density of DNA— 10 mg/ml (bacteria), 100 mg/ml (eukaryotic), 500 mg/ml (T 4 head)

Figure 09. 01: The length of nucleic acid is much greater than the dimensions of the surrounding compartment.

9. 2 Viral Genomes Are Packaged into Their Coats • Packing mechanism • • 1) protein shells are assembled around nucleic acid 2) capsid forms DNA packing • capsid – The external protein coat of a virus particle. • The length of DNA that can be incorporated into a virus is limited by the structure of the headshell. • Nucleic acid within the headshell is extremely condensed. Figure 09. 02: A helical path for TMV RNA is created by the stacking of protein subunits in the virion.

TMV virus packing (capsid protein-RNA associated) 1. Duplex hairpin in RNA (nucleation center) 2. Capsid proteins assembled the RNA bidirections up to RNA end Figure 09. 02: A helical path for TMV RNA is created by the stacking of protein subunits in the virion.

• Filamentous RNA viruses condense the RNA genome as they assemble the headshell around it. • nucleation center – A duplex hairpin in TMV (tobacco mosaic virus) in which assembly of coat protein with RNA is initiated.

9. 2 Viral Genomes Are Packaged into Their Coats-Lambda Phage Head structure DNA packing Head shell core Two step: translocation and condensation Translocation ATP-dependent mechanism Rolling circle model Figure 09. 03: Maturation of phage lambda passes through several stages. Top photo reproduced from Cue, D. and Feiss, M. , Proc. Natl. Acad. Sci. USA 90 (1993): 9290 -9294. Copyright 1993 National Academy of Science, USA. Photo courtesy of Michael G. Feiss, University of Iowa. Bottom photo courtesy of Robert Duda, University of Pittsburgh.

Figure 09. 04: Terminase protein binds to specific sites on a multimer of virus genomes generated by rolling circle replication.

• Spherical DNA viruses insert the DNA into a preassembled protein shell. • terminase – An enzyme that cleaves multimers of a viral genome and then uses hydrolysis of ATP to provide the energy to translocate the DNA into an empty viral capsid starting with the cleaved end. • Cos site

9. 3 The Bacterial Genome Is a Supercoiled Nucleoid • The bacterial nucleoid is ~80% DNA by mass and can be unfolded by agents that act on RNA or protein. • protein and RNA are essential for genome structure maintaining • The proteins that are responsible for condensing the DNA have not been identified. Genomes are organized in definite bodies (compact clump; occupied 1/3 volume) Figure 09. 05: E. coli bacterium, colored transmission electron micrograph (TEM). © Dr. Klaus Boller/Photo Researchers, Inc.

DNA intercalating agent; Et-Br Generating positive supercoil in closed circular DNA Open DNA, tension is reduced by rotation E. coli, negative supercoiled , domain Figure 09. 06: The nucleoid spills out of a lysed E. coli cell in the form of loops of a fiber. © G. Murti/Photo Researchers, Inc.

• The nucleoid has ~400 independent negatively supercoiled domains. • 400 domain/10 kb • The average density of supercoiling is ~1 supercoil/100 bp. Figure 09. 07: The bacterial genome consists of a large number of loops of duplex DNA (in the form of a fiber), each of which is secured at the base to form an independent structural domain.

Figure 09. 08: An unconstrained supercoil in the DNA path creates tension, but no tension is transmitted along DNA when a supercoil is constrained by protein binding.

9. 4 Eukaryotic DNA Has Loops and Domains Attached to a Scaffold • DNA of interphase chromatin is negatively supercoiled into independent domains of ~85 kb. • Metaphase chromosomes have a protein scaffold to which the loops of supercoiled DNA are attached. Figure 09. 09: Histone-depleted chromosomes consist of a protein scaffold to which loops of DNA are anchored.

• DNA is attached to the nuclear matrix at specific sequences called MARs or SARs. • The MARs are A-T-rich but do not have any specific consensus sequence. • metaphase ( or mitotic) scaffold – A proteinaceous structure in the shape of a sister chromatid pair, generated when chromosomes are depleted of histones.

9. 5 Chromatin Is Divided into Euchromatin and Heterochromatin Figure 09. 10: The sister chromatids of a mitotic pair each consist of a fiber (~30 nm in diameter) compactly folded into the chromosome. © Biophoto Associates/Photo Researchers, Inc.

Figure 09. B 01 Adapted from an illustration by Darryl Leja, National Human Genome Research Institute (www. genome. gov).

Figure 09. B 02 A: Spectral karyotyping (SKY), an application of chromosome painting. Photo courtesy of Johannes Wienberg, Ludwig. Maximilians-University, and Thomas Ried, National Institutes of Health.

Figure 09. B 02 B: Photo courtesy of Johannes Wienberg, Ludwig. Maximilians-University, and Thomas Ried, National Institutes of Health.

9. 5 Chromatin Is Divided into Euchromatin and Heterochromatin • Individual chromosomes can be seen only during mitosis. • During interphase, the general mass of chromatin is in the form of euchromatin, which is slightly less tightly packed than mitotic chromosomes. • Regions of heterochromatin remain densely packed throughout interphase. Figure 09. 11: A thin section through a nucleus stained with Feulgen shows • chromocenter – An aggregate of heterochromatin as compact regions heterochromatin from different clustered near the nucleolus and chromosomes. nuclear membrane. • Constitutive and facultative heterochromatin Photo courtesy of Edmond Puvion, Centre National de la Recherche Scientifique

Common features of heterochromatin 1. 2. 3. 4. 5. Permanently condensed Late replication and low recombination Multi-repeated sequence and very low or absent transcription Gene is rare Translocation into this region induce silencing (exception; ribosomal DNA) For active transcription, location of euchromatin is necessary but not sufficient

9. 6 Chromosomes Have Banding Patterns • Certain staining techniques cause the chromosomes to have the appearance of a series of striations, which are called Gbands. • The bands are lower in G-C content than the interbands. • Genes are concentrated in the G-C -rich interbands. Figure 09. 13: The human X chromosome can be divided into distinct regions by its banding pattern.

Figure 09. 12: G-banding generates a characteristic lateral series of bands in each member of the chromosome set. Photo courtesy of Lisa Shaffer, Washington State University-Spokane

Figure 09. 13: The human X chromosome can be divided into distinct regions by its banding pattern.

Chromosome 12 Contains over 1600 genes Contains over 130 million base pairs, of which over 95% have been determined See the diseases associated with chromosome 12 in the Map. Viewer.

Phenylketonuria (PKU) is an inherited error of metabolism caused by a deficiency in the enzyme phenylalanine hydroxylase. Loss of this enzyme results in mental retardation, organ damage, unusual posture and can, in cases of maternal PKU, severely compromise pregnancy. Classical PKU is an autosomal recessive disorder, caused by mutations in both alleles of the gene for phenylalanine hydroxylase (PAH), found on chromosome 12. In the body, phenylalanine hydroxylase converts the amino acid phenylalanine to tyrosine, another amino acid. Mutations in both copies of the gene for PAH means that the enzyme is inactive or is less efficient, and the concentration of phenylalanine in the body can build up to toxic levels. In some cases, mutations in PAH will result in a phenotypically mild form of PKU called hyperphenylalanemia. Both diseases are the result of a variety of mutations in the PAH locus; in those cases where a patient is heterozygous for two mutations of PAH (ie each copy of the gene has a different mutation), the milder mutation will predominate. A form of PKU has been discovered in mice, and these model organisms are helping us to better understand the disease, and find treatments against it. With careful dietary supervision, children born with PKU can lead normal lives, and mothers who have the disease can produce healthy children

Chromosome 15 Contains approximately 1200 genes Contains approximately 100 million base pairs, of which over 80% have been determined See the diseases associated with chromosome 15 in the Map. Viewer.

Prader-Willi syndrome (PWS) is an uncommon inherited disorder characterized by mental retardation, decreased muscle tone, short stature, emotional lability and an insatiable appetite which can lead to life -threatening obesity. The syndrome was first described in 1956 by Drs. Prader, Labhart, and Willi. PWS is caused by the absence of segment 11 -13 on the long arm of the paternally derived chromosome 15. In 70 -80% of PWS cases, the region is missing due to a deletion. Certain genes in this region are normally suppressed on the maternal chromosome, so, for normal development to occur, they must be expressed on the paternal chromosome. When these paternally derived genes are absent or disrupted, the PWS phenotype results. When this same segment is missing from the maternally derived chromosome 15, a completely different disease, Angelman syndrome, arises. This pattern of inheritance when expression of a gene depends on whether it is inherited from the mother or the father is called genomic imprinting. The mechanism of imprinting is uncertain, but, it may involve DNA methylation. Genes found in the PWS chromosomal region code for the small ribonucleoprotein N (SNRPN). SNRPN is involved in m. RNA processing, an intermediate step between DNA transcripton and protein formation. A mouse model of PWS has been developed with a large deletion which includes the SNRPN region and the PWS 'imprinting centre' (IC) and shows a phenotype similar to infants with PWS. These and other molecular biology techniques may lead to a better understanding of PWS and the mechanisms of genomic imprinting

Angelman syndrome (AS) is an uncommon neurogenetic disorder characterized by mental retardation, abnormal gait, speech impairment, seizures, and an inappropriate happy demeanor that includes frequent laughing, smiling, and excitability. The uncoordinated gait and laughter have caused some people to refer to this disorder as the "happy puppet" syndrome. The genetic basis of AS is very complex, but the majority of cases are due to a deletion of segment 15 q 11 q 13 on the maternally derived chromosome 15. When this same region is missing from the paternally derived chromosome, an entirely different disorder, Prader Willi syndrome, results. This phenomenon when the expression of genetic material depends on whether it has been inherited from the mother or the father is termed genomic imprinting. The ubiquitin ligase gene (UBE 3 A) is found in the AS chromosomal region. It codes for an enzyme that is a key part of a cellular protein degradation system. AS is thought to occur when mutations in UBE 3 A disrupt protein break down during brain development. In a mouse model of AS, affected animals had much less maternally inherited UBE 3 A than their unaffected litter mates. However, this difference in UBE 3 A levels was only found in the hippocampus and the cerebellum, and not all of the brain. This animal model and other molecular techniques are helping us learn

9. 7 Polytene Chromosomes Form Bands That Expand at Sites of Gene Expression Figure 09. 14: The polytene chromosomes of D. melanogaster form an alternating series of bands and interbands. Photo courtesy of José Bonner, Indiana University • chromomeres – Densely staining granules visible in chromosomes under certain conditions, especially early in meiosis, when a chromosome may appear to consist of a series of chromomeres. • polytene chromosomes – Chromosomes that are generated by successive replications of a chromosome set without separation of the replicas.

Some specialized eukaryotic cells increase cell volume via endomitosis, where DNA synthesis is repeated without cell division and a normal chromosome develops into a giant polytene chromosome results Figure 09. 14: The polytene chromosomes of D. melanogaster form an alternating series of bands and interbands. Photo courtesy of José Bonner, Indiana University

in situ hybridization – Hybridization performed by denaturing the DNA of cells squashed on a microscope slide so that reaction is possible with an added single-stranded RNA or DNA; the added preparation is radioactively labeled and its hybridization is followed by autoradiography. Figure 09. 15: A magnified view of bands 87 A and 87 C shows their hybridization in situ with labeled RNA extracted from heat-shocked cells. Photo courtesy of José Bonner, Indiana University

• Bands that are sites of gene expression on polytene chromosomes expand to give “puffs. ” Figure 09. 16: Displayed is a small segment of chromosome 3 before (top) and after (bottom) heat shock. Chromosomes are stained for DNA (blue) and for Pol II (green). Photo courtesy of Victor G. Corces, Emory University

9. 8 The Eukaryotic Chromosome Is a Segregation Device • • A eukaryotic chromosome is held on the mitotic spindle by the attachment of microtubules to the kinetochore that forms in its centromeric region. microtubule organizing center (MTOC) – A region from which microtubules emanate. – In animal cells the centrosome is the major microtubule organizing center. centromere – A constricted region of a chromosome that includes the site of attachment (the kinetochore) to the mitotic or meiotic spindle. It may consist of unique DNA sequences or highly repetitive sequences and proteins not found anywhere else in the chromosome. acentric fragment – A fragment of a chromosome (generated by breakage) that lacks a centromere and is lost at cell division.

Figure 09. 17: Chromosomes are pulled to the poles via microtubules that attach at the centromeres.

Figure 09. 18: The centromere is identified by a DNA sequence that binds specific proteins.

Adapted from Y. Datal et al. , Proc. Natl. Acad. Sci. USA 104 (2007): 15974– 15981.

9. 9 Regional Centromeres Contain a Centromeric Histone H 3 Variant and Repetitive DNA • Centromeres are characterized by a centromere-specific histone H 3 variant and often contain heterochromatin that is rich in satellite DNA sequences. • The function of the repetitive DNA is not known. • Satellite DNA-171 Bp repeated Figure 09. 19: A model of the overall structure of a regional centromere.

9. 10 Point Centromeres in S. cerevisiae Contain Short, Essential Protein-Binding DNA Sequences • CEN elements are identified in S. cerevisiae by the ability to allow a plasmid to segregate accurately at mitosis. • CEN elements consist of the short, conserved sequences CDE-I and CDE-III that flank the A-T-rich region CDE-II. Figure 09. 20: Three conserved regions can be identified by the sequence homologies between yeast CEN elements.

9. 10 Point Centromeres in S. cerevisiae Contain Short, Essential Protein-Binding DNA Sequences Figure 09. 21: The DNA at CDE-II is wound around a protein aggregate including Cse 4 p, CDE-III is bound to CBF 3 and CDE-I is bound to CBF 1. • A specialized protein complex containing the histone variant Cse 4 is formed at CDE-II. • The CBF 3 protein complex that binds to CDE-III is essential for centromeric function. • The proteins that bind CEN serve as an assembly platform for the kinetochore and provide the connection to microtubules.

9. 11 Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends • The telomere is required for the stability of the chromosome end. • A telomere consists of a simple repeat where a C+A-rich strand has the sequence C>1(A/T)1– 4. Figure 09. 22: A typical telomere has a simple repeating structure with a G-T-rich strand that extends beyond the C-A-rich strand.

Figure 10. 11 The Biology of Cancer (© Garland Science 2007)

Figure 10. 13 a The Biology of Cancer (© Garland Science 2007)

Figure 09. 23: The crystal structure of a short repeating sequence from the human telomere forms three stakced G quartets.

Figure 09. 24: Colored transmission electron micrograph (TEM) of a telomere. © Dr. Gopal Murti/Photo Researchers, Inc.

Figure 10. 17 a The Biology of Cancer (© Garland Science 2007)

• The protein TRF 2 catalyzes a reaction in which the 3′ repeating unit of the G+T-rich strand forms a loop by displacing its homolog in an upstream region of the telomere. Figure 09. 25: The 3' single-stranded end of the telomere (TTAGGG)n displaces the homologous repeats from duplex DNA to form a t-loop. The reaction is catalyzed by TRF 2.

• Telomerase uses the 3′-OH of the G+T telomeric strand to prime synthesis of tandem TTGGGG repeats. • The RNA component of telomerase has a sequence that pairs with the C+A-rich repeats. • One of the protein subunits is a reverse transcriptase that uses the RNA as template to synthesis the G+T-rich sequence. Figure 09. 27: Telomerase positions itself by base pairing between the RNA template and the protruding single-stranded DNA primer.

Figure 10. 18 The Biology of Cancer (© Garland Science 2007)

Figure 10. 19 The Biology of Cancer (© Garland Science 2007)

9. 11 Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends • Telomerase is expressed in actively dividing cells and is not expressed in differentiated cells. • Loss of telomeres results in senescence. • Escape from senescence can occur if telomerase is reactivated, or via unequal homologous recombination to restore telomeres.

9. 11 Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends Figure 09. 28: Mutation in telomerase causes telomeres to shorten in each cell division. Eventual loss of the telomere causes chromosome breaks and rearrangements.

Figure 10. 20 The Biology of Cancer (© Garland Science 2007)

Figure 10. 21 a The Biology of Cancer (© Garland Science 2007)

Figure 10. 31 The Biology of Cancer (© Garland Science 2007)

Dyskerin-h. TR X-linked dyskeratosis congenia Figure 10. 32 a The Biology of Cancer (© Garland Science 2007)

Figure 09. 22: A typical telomere has a simple repeating structure with a G-T-rich strand that extends beyond the C-A-rich strand.

Figure 09. FTR 01 A: FISH, Chromosome Painting, and Spectral Karyotyping

Figure 09. FTR 01 B: FISH, Chromosome Painting, and Spectral Karyotyping

1. Senescence Progeroid disorders; prematured senescence Human progeroid diseases 1. 2. 3. 4. Werner syndrome Hutchinson-Gilford Syndrome Ataxia-Telangiactia Cockayne syndrome HGS Trigger of senescence 1. Oxidative stress 2. IGF 3. Energy balance 4. replication Cockayne syndrome

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