#2 Lecture2

Chapter 9

Chromosomes

9.1. Introduction

  Cellular genetic material (genome): DNA or RNA •  They must be condensed to be packaged into phages, viruses,

bacteria, or eukaryotic nuclei. •  Genome condensation results from its binding to basic proteins. •  Bacterial genome is present as nucleoid. •  Eukaryotic genome is present as chromatin at interphase or

chromosome during mitosis.

Figure 9.1

•  Chromatin: the state of DNA and its associated proteins during the interphase of the eukaryotic cell cycle (G1-S-G2)

•  Chromosome: a discrete unit of the genome, only visible during mitosis (M phase).

Eukaryotic cell cycle 9.1. Introduction

Mitosis movie

M phase

9.2 Viral Genomes Are Packaged into Their Coats

•  Each virus has an inner part consisting of a nucleic acid core

•  Wrapped around it is an outer protein coat – capsid

•  The length of genome that can be incorporated into a virus is limited by the structure of the capsid.

•  Nucleic acid (both DNA and RNA) within a capsid is extremely condensed.

  Filamentous RNA viruses condense the RNA genome as they assemble the capsid around it.

  The best-characterized example is tobacco mosaic virus (TMV).

•  Bidirectional assembly from nucleation center (secondary RNA structure).

•  The capsid proteins are stacked in a circle of 17 protein subunits and forms a helix along the RNA; RNA becomes coiled in a helical array on the inside of the protein shell.


Figure 9.2

  Spherical DNA viruses (e.g., Phage lambda and T4)

  Phage lambda assembly • Empty protein core (prohead I) is made. • Core changes its shape to prohead II. • DNA packaging begins and head

expands while maintaining its shape. •  The headshell reaches full size and

sealed by the addition of the tail.


Figure 9.3

  Lambda DNA packaging involves two types of reaction: translocation and condensation.

•  Translocation – terminase enzyme cleaves multimeric λ DNA into a monomer and translocates the DNA into an empty capsid using ATP hydrolysis (energy).

•  Condensation - DNA is condensed as it enters the head. Its mechanism is unknown but it is not sequence-specific.


Figure 9.4

9.3. Bacterial Genome Is a Supercoiled Nucleoid

  Nucleoid

•  “Nucleus-like”, genetic material of bacteria, no membrane.

•  Looped DNA is released when bacteria are lysed and DNA is bound to proteins. The proteins that are responsible for condensing the DNA have not been identified.

•  The bacterial nucleoid is ~80% DNA by mass (eukaryotes, ~50%)

• Unfolded by RNase or proteinase; RNA and protein are important to maintain nucleoid structure

Figure 9.5

Figure 9.6

  The nucleoid has ~400 independent negatively supercoiled domains in E. Coli genome.

  Each loop contains ~10-40 kb DNA and secured at base by unknown mechanism.

  Different degrees of supercoiling in different regions (domains) of the genome – supercoiling is important in transcription.

•  If a supercoiled DNA is unconstrained, negative supercoils generate a state of torsional tension that is transmitted freely along the DNA within a domain. The DNA is in a dynamic equilibrium between the states of tension and unwinding.

•  If supercoiling is constrained by binding proteins, supercoiling is sustained and no tension is transmitted along the molecule.

9.3. Bacterial Genome Is a Supercoiled Nucleoid

Figure 9.7

Figure 9.8

9.4. Eukaryotic DNA Has Loops and Domains Attached to a Scaffold

•  Eukaryotic DNA has domains (~85 kb/domain)

•  Loops can be seen when the majority of proteins (i.e., histones) are extracted from metaphase chromosomes.

•  Metaphase scaffold contains ~8% of the original protein content and resembles the general form of sister chromatids.

•  In interphase nuclei, this proteinaceous structure changes its organization to occupy the entire nucleus - matrix rather than scaffold

•  Specific DNA regions are attached to the nuclear matrix or scaffold: matrix attachment regions (MARs) or scaffold attachment regions (SARs).

•  The MARs are A-T-rich (~70%) but do not have any specific consensus sequence.

Figure 9.9

9.5. Chromatin is Divided into Euchromatin and Heterochromatin

•  Chromatin: the state of nuclear DNA and proteins during interphase (chromosome during mitosis).

•  During the most of cell cycle (i.e., interphase), eukaryotic DNA (chromatin) occupies the nucleus in which individual chromosomes cannot be distinguished; they can be seen only during mitosis.

•  DNA is 5-10 times more condensed in mitotic chromosomes than in interphase chromatin.

•  Chromatin can be divided into two types of material, euchromatin and heterochromatin.

Sister chromatids at metaphase (EM image)

Figure 9.10

Centromere

  Euchromatin and Heterochromatin

•  During interphase, most of the nuclear region is occupied by euchromatin, which is (1) less tightly packed than mitotic chromosomes, (2) stains lightly with DNA-specific dyes, and (3) contains transcriptionally active genes.

•  Regions of heterochromatin remain densely packed throughout interphase. They strongly stains with DNA dyes and are as dense as chromosome.


Figure 9.11

−  Found at several locations within the chromatin, including the centromeres. −  Chromocenter: aggregate of various heterochromatic regions −  Constitutive chromatin: regions that are always heterochromatic. −  Facultative chromatin: euchromatins converted to a heterochromatic state

  Features of Constitutive Heterochromatin

•  Permanently condensed •  Multiple repeats of a few sequences of DNA that are not

transcribed or transcribed at very low levels •  Replicates late in S phase and reduced frequency of

recombination •  Reduced density of genes compared with euchromatin •  Genes translocate into or near it are often inactivated (ribosomal DNA is exception; heterochromatic but

transcriptionally active)


9.6. Chromosomes Have Banding Patterns •  Certain staining techniques (e.g., Giemsa)

cause the chromosomes to have the appearance of a series of striations, which are called G-bands.

•  Mechanism – unknown

•  Each band is ~107 bp.

•  The bands are lower in G-C content than the interbands.

•  Genes are concentrated in interbands.

•  Each chromosome has a unique pattern. -  Identify chromosomes, translocation

between chromosomes.

Figure 9.12

Figure 9.13

•  A gene location is indicated by its position on the long (q) or short (p) arm, region of the arm, band, and subband. − CFTR is located at 7q31.2.

Methods and Techniques

  Fluorescent in situ hybridization (FISH) utilizes probes that bind to a specific DNA sequence within the chromosome. Classic staining (e.g., G-bands) does not detect small changes (translocation, deletions, insertions) involving small segments within a chromosome. Its application includes identifying chromosomal aberrations and locating genes on a chromosome and chromatin.

Figure B9.1

Methods and Techniques

  Improvement of FISH for more robust analysis

•  Chromosome painting: use multiple fluorescent probes binding all along a particular chromosome (visualize one chromosome), unable to see all 23 chromosomes in a unique color because there are not enough fluorescent dyes with sufficient color differences to mark all 23 chromosomes in a unique color.

•  Spectral karyotyping (SKY): simultaneously visualize all pairs of chromosomes in different colors. Chromosome-specific probes are labeled with different assortment of colors. Analyzed and presented with pseudo colors to each different color combination.

Figure B9.2

•  Polytene chromosomes are found in the interphase nuclei of specific tissues of the larvae of dipteran (two-winged) insects such as Drosophila melanogaster. They possess increased diameter and greater length compared to their usual condition.

•  Polytene chromosomes are generated by successive replications of a chromosome without separation of sister chromatids (endoreduplication): can duplicate up to a DNA content x1024 of a individual chromosome.

•  They are composed of bands and interbands (note that bands and interbands here are different from G-bands).

•  Bands contain most of the mass of DNA and are intensely stained; but interbands are lightly stained

9.7. Polytene Chromosomes Form Bands That Expand at Sites of Gene Expression

Figure 9.14

•  Sites of active transcription can be visualized: bands that are sites of gene expression (i.e., transcription) on polytene chromosomes expand to give “puffs”.

•  During larval development, puffs appear and regress in temporal and tissue-specific patterns; many puffs are induced by ecdysone (fly horhormone).

•  Puffs are useful to understand gene function.

RNA Polymerase II •  Puffing attracts proteins

associated with transcription.

•  Heat shock reveals regions containing heat shock response genes.

before

9.7. Polytene Chromosomes Form Bands That Expand at Sites of Gene Expression

Figure 9.16

after

9.8. The Eukaryotic Chromosome Is a Segregation Device

•  Spindle: microtubules that guide movements of chromosomes •  Microtubule organizing centers (MTOCs) – a region from which

microbubules emanate (centrosome in animal cells) •  Centromere: a constricted region of a chromosome that includes the site

of attachment to the mitotic spindle. It is heterochromatin and repetitive DNA sequence.

•  Kinetochore: a protein complex on centromere that attaches the chromosome to the mitotic spindle.

•  Cohesins: protein complexes that hold two sister chromatids together. They are degraded before segregation.

Figure 9.17

Mitosis Movie

9.9. Regional Centromeres Contain a Centromeric Histone H3 Variant and Repetitive DNA

•  Centromere is not defined by DNA sequences and instead by chromatin structure (Chap. 10); chromatin is composed of DNA and histones (H1, H2A, H2B, H3, and H4).

•  Centromere is specified by centromere-specific histone H3 (CENP-A/CenH3); CenH3-containing nucleosomes (chromatin subunit) protrude from the bulk chromatin.

•  The length of DNA required for centromeric function is often long; one exception is Saccharomyces cerevisiae (~120 bp). - S. pombe: 40 – 100 kb - Drosophila: 200 – 600 kb - Arabidopsis: >500 kb

Figure 9.19

•  The sequences required for centromeric function fall within ~120 bp (CEN region) provides a useful tool to identify proteins that bind centromere and form kinetochore.

•  Every chromosome of S. cerevisiae has a CEN region. Fragments from different chromosomes are interchangeable; CEN does not distinquish one chromosome from another.

•  Three cell cycle-dependent elements (CDEs) in the CEN region: - CDE-I: 9bp, highly conserved. - CDE-II: >90% A-T rich sequence of 80-90 bp, its function depends on the length rather than its exact sequence. - CDE-III; 11bp, highly conserved, some mutations are tolerated but CCG are essential.

9.10. Point Centromeres in S. cerevisiae Contain Short, Essential Protein-Binding DNA Sequences

Figure 9.20. The S. cerevisiae centromere has a long A-T stretch flanked by short conserved sequences.

9.10. Point Centromeres in S. cerevisiae Contain Short, Essential Protein-Binding DNA Sequences

Figure 9.21

- Protein Structure at CEN Connecting Chromosomes to Microtubules

•  CEN recruits 3 DNA-binding proteins, Cbf1, Cbf3 and Mif2 (CENP-C in muticellular eukaryotes).

•  A specialized chromatin structure by binding CDE-II to Cse4 (the yeast CenH3 histone variant). Scm3 is required for association of Cse4 with CEN.

•  CDE-I binds Cbf1 homodimer •  CDE-III binds Cbf3 (four-protein complex) •  These proteins interact with other proteins

(Ctf19, Mcm21, Okp1) that link the centromeric complex to the kinetochore proteins (~70 proteins identified in yeast) and to the microtubule

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

•  The telomere is required for the stability of the eukaryotic chromosome (linear DNA).

•  The cell must be able to distinguish the telomere (stable) from free DNA ends caused by damage.

•  A long series of short, tandemly repeated sequences (100 -1000 repeats).

•  A telomere consists of a simple repeat, Cn(A/T)m; n>1, m is 1 to 4.

•  Highly conserved among eukaryotes.

•  Extension of 3’ overhang G-T-rich strand.

Figure 9.22

•  Guanine bases have an unusual ability to form hydrogen bonds with one another single-stranded G-rich tail in telomere can form “quartets” of G residues.

•  Each quartet contains 4 Gs and forms a planar structure.


Figure 9.23

•  A loop of DNA forms at the telomere.

5~10 kb


Figure 9.24

•  Single-stranded (TTAGGG)n displaces the same sequence in an upstream of the telomere, resulting in D-loop like structure.

•  The protein Trf2 catalyzes loop formation. •  Deletion of Trf2 causes chromosome

rearrangements. •  Shelterin: complex of 6 telomeric proteins

(Trf1, Trf2, Rap1, Tin2, Tpp1 and Pot1)

Figure 9.25

•  Telomere has the ability to be extended, by addition of telomeric repeats to the end of the chromosome in every replication cycle.

•  Telomerase uses the 3′–OH of the G + T telomeric strand to prime synthesis of tandem TTGGGG repeats.

•  The RNA component (150~1300 nt) of telomerase has a sequence of 15~22 nt identical to two repeats of the C+A-rich repeating sequence.

•  One of the protein subunits is a reverse transcriptase that uses the RNA as template to synthesize the G + T-rich DNA sequence.

•  Discontinuous synthesis. •  Telomere length (5~15 kb in human and ~300

bp in yeast) is determined by Est1 and Est3 in yeast but not by telomerase.


Telomere Extension by Telomerase Figure 9.27

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