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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
9.1. Introduction
! 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).
Mitosis movie
M phase
! 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.
Figure 9.2
Figure 9.3
Figure 9.4
Centromere
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
Figure B9.1
Figure B9.2
9.7. Polytene Chromosomes Form Bands That Expand at Sites of Gene Expression
! 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
Figure 9.14
9.7. Polytene Chromosomes Form Bands That Expand at Sites of Gene Expression
! 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). Figure 9.16 ! 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 after
Figure 9.17
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 centromerespecific 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
9.10. Point Centromeres in S. cerevisiae Contain Short, Essential Protein-Binding DNA Sequences
! 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.
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
- 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
Figure 9.21
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
9.11. Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends
! 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
9.11. Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends
! A loop of DNA forms at the telomere. ! 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
9.11. Telomeres Have Simple Repeating Sequences That Seal the Chromosome Ends
Telomere Extension by Telomerase
! 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.
Figure 9.27