Chapter 9 Chromosomes

9.1 Introduction 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.

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.

9.2 Viral Genomes Are Packaged into Their Coats 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.

9.2 Viral Genomes Are Packaged into Their Coats 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. 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): 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.

9.2 Viral Genomes Are Packaged into Their Coats 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. Figure 09.04: Terminase protein binds to specific sites on a multimer of virus genomes generated by rolling circle replication.

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. The proteins that are responsible for condensing the DNA have not been identified. Figure 09.05: E. coli bacterium, colored transmission electron micrograph (TEM). © Dr. Klaus Boller/Photo Researchers, Inc.

9.3 The Bacterial Genome Is a Supercoiled Nucleoid The nucleoid has ~400 independent negatively supercoiled domains. The average density of supercoiling is ~1 supercoil/100bp. 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.

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.

9.4 Eukaryotic DNA Has Loops and Domains Attached to a Scaffold 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 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 heterochromatin as compact regions clustered near the nucleolus and nuclear membrane. Photo courtesy of Edmond Puvion, Centre National de la Recherche Scientifique

9.5 Chromatin Is Divided into Euchromatin and Heterochromatin chromocenter – An aggregate of heterochromatin from different chromosomes.

9.6 Chromosomes Have Banding Patterns Certain staining techniques cause the chromosomes to have the appearance of a series of striations, which are called G-bands. 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.

9.7 Polytene Chromosomes Form Bands That Expand at Sites of Gene Expression 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. Figure 09.14: The polytene chromosomes of D. melanogaster form an alternating series of bands and interbands. Photo courtesy of José Bonner, Indiana University

9.7 Polytene Chromosomes Form Bands That Expand at Sites of Gene Expression Polytene chromosomes of dipterans have a series of bands that can be used as a cytological map. 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.

9.7 Polytene Chromosomes Form Bands That Expand at Sites of Gene Expression Bands that are sites of gene expression on polytene chromosomes expand to give “puffs.” Figure 09.15: A magnified view of bands 87A and 87C shows their hybridization in situ with labeled RNA extracted from heat- shocked cells. 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 José Bonner, Indiana University 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.

9.8 The Eukaryotic Chromosome Is a Segregation Device 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.

9.8 The Eukaryotic Chromosome Is a Segregation Device 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.

9.9 Regional Centromeres Contain a Centromeric Histone H3 Variant and Repetitive DNA Centromeres are characterized by a centromere-specific histone H3 variant and often contain heterochromatin that is rich in satellite DNA sequences. The function of the repetitive DNA is not known.

9.13 Regional Centromeres Contain a Centromeric Histone H3 Variant and Repetitive DNA Figure 09.19: A model of the overall structure of a regional centromere. Adapted from Y. Datal et al., Proc. Natl. Acad. Sci. USA 104 (2007): 15974–15981.

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 A specialized protein complex containing the histone variant Cse4 is formed at CDE-II. The CBF3 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. Figure 09.21: The DNA at CDE-II is wound around a protein aggregate including Cse4p, CDE-III is bound to CBF3 and CDE-I is bound to CBF1.

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.

9.11 Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends The protein TRF2 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 TRF2.

9.11 Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends 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.

9.11 Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends Figure 09.27: Telomerase positions itself by base pairing between the RNA template and the protruding single-stranded DNA primer.

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.