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Welcome Each of You to My Molecular Biology Class

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Title: Welcome Each of You to My Molecular Biology Class


1
Welcome Each of You to My Molecular Biology Class
2
Molecular Biology of the Gene, 5/E --- Watson et
al. (2004)
Part I Chemistry and Genetics Part II
Maintenance of the Genome Part III Expression
of the Genome Part IV Regulation Part V Methods
3/011/05
3
Part II Maintenance of the Genome
Dedicated to the structure of DNA and the
processes that propagate, maintain and alter it
from one cell generation to the next
4
Ch 6 The structures of DNA and RNA Ch 7
Chromosomes, chromatins and the nucleosome Ch 8
The replication of DNA Ch 9 The mutability and
repair of DNA Ch 10 Homologous recombination at
the molecular level Ch 11 Site-specific
recombination and transposition of DNA
3/11/05
5
  • CHAPTER 7 Chromosomes, chromatin, and the
    nucleosome

6
Consider the structure of DNA within the cell,
and the biological relevance of the structure.
7
Vocabulary
Nucleus ??? Nucleolus ?? Nucleoid ?? Mitosis
????Meiosis???? interphase???? Histone
???Nucleosome ??? Chromotasome
???? Chromosome ??? Chromatin ???eu-
hetero- Centromere (???) Telomere(??) Repetitive
DNA (??DNA) Tandem gene cluster(???? ?)
8
OUTLINE
  • Chromosome sequence diversity
  • Chromosome duplication segregation
  • The nucleosome
  • Higher-order chromatin structure
  • Regulation of chromatin structure
  • Nucleosome assembly

9
Prokaryotic and eukaryotic chromosome structure
D1 Prokaryotic chromosome (?? ???)
10
D1 Prokaryotic chromosome structure
D1-1 The E. coli chromosome
  1. A single closed-circular DNA, 4.6 X 106bp
  2. The DNA packaged into a region known as Nucleoid
    (??) that contains high concentration of DNA (up
    to 30-50 mg/ml) as well as all proteins
    associated with DNA.
  3. Continuous replication (no cell cycle)
  4. Attach to a part of cell membrane by ?

11
D1 Prokaryotic chromosome structure
Nucleoid (??,??) Bacterial chromosome ?????
12
D1 Prokaryotic chromosome structure
D1-2 DNA domains/loops
  1. Observed under electron microscope
  2. 50-100 domains or loops per E. coli chromosome,
    with 50-100 kb/loop
  3. The ends of loops are constrained (??) by
  4. Are loops dynamic or static? What do you think,
    why? Could you design experiments to prove it?

13
D1 Prokaryotic chromosome structure
Domain/ Loop
Basic protein
Member binding proteins?
Supercoiled DNA
14
D4 Genome Complexity
D1 Prokaryotic chromosome structure
Supercoiled domain
Non-supercoiled domain
Protein-membrane core or scaffold
15
D1 Prokaryotic chromosome structure
D1-3 Supercoiling of the genome
  • E. coli chromosome as a whole is negatively
    supercoiled
  • Individual domains may be supercoiled
    independently (topological independent) because
    the protein-membrane scaffold may prevent DNA
    rotation.
  • Oops!Direct biochemical evidence is lacking for
    different level of supercoiling in different
    domains. Do you want to try an experiment?

back
16
D1 Prokaryotic chromosome structure
D1-4 DNA-binding proteins
back
  • Histone-like proteins essential for DNA packaging
    to stabilize and constrain the supercoiling.
  • HU a small basic dimeric (????) protein,
    non-specific binding to DNA, most abundant.
  • H-NS (protein H1) neutral monomeric (????),
    partially non-specific binding
  • Site-specific DNA binding proteins important for
    organization of DNA domains (RNA polymerases, IHF
    etc).

17
Supplementary 1
  • Organelle genomes
  • circular, multiple copies
  • ctDNA chloroplast (???) DNA, 140kb in plants,
    and lt200kb in lower eukaryotes. Similar to
    cyanobacteria (???)
  • mtDNA mitochondrial (???) DNA, 16.5 kb in
    mammals, 80 kb in yeast, gt100kb in plant. Similar
    to ?-purple bacteria, Rickettsia)

mtDNA
18
Supplementary 2
Range of genome size found in different organism
phyla.
19
Prokaryotic and eukaryotic chromosome structure
D2. Chromatin Structure (?????)
20
D2 Chromatin structure
D2-1. Chromatin (???)
A highly organized complex of DNA and protein
(nucleoprotein complex), which makes up the
eukaryotic chromosomes (???). gt50 of the mass is
protein
21
D2 Chromatin structure
Chromatin structure enables the chromosomes to
alter their compactness as the cell progress the
cell cycle.
Mitosis
Interphase
Chromatin (diffused)
Chromosome (condensed)
22
D2 Chromatin structure
D2-23. Histones (???) and Nucleosomes (???)
  1. The major protein components of chromatin
  2. Four families of core histone H2A, H2B, H3 and
    H4. An additional non-core histone H1
  3. Small, 10 kDa for core histones and 23 kDa for
    H1.
  4. Basic (rich in lysine and arginine) and tightly
    binds to DNA

23
D2 Chromatin structure
Members of the same histone class are very
highly conserved between unrelated species, but
there is not much similarity in sequence between
the different histone class (page 50 of your text
book). What does this fact suggest to you?
24
D2 Chromatin structure
Histone octamer (??????)
Top view
Nucleosome core
Side view
25
D2 Chromatin structure
DNA
Histone octamer
Nucleosome core 146 bp, 1.8 superhelical turn
Histone H1
Chromatosome 166 bp, 2 superhelical turn
26
D2 Chromatin structure
D2-4. The role of H1
  1. Stabilizes the point at which DNA enters and
    leaves the nucleosome core.
  2. C- tail of H1 stabilizes the DNA between the
    nucleosome cores.
  • 23 kDa, located outside of nucleosome core, binds
    to DNA more loosely
  • Less conserved in its sequence (What does this
    suggests?)

27
D2 Chromatin structure
D2-5. Linker DNA
The additional DNA required to make up the 200 bp
nucleosomal repeat, 55 bp
How was the linker identified?
28
Beads on a string structure
Nucleosome repeat Core linker DNA 200 bp
Histone H1
Nucleosome
Linker DNA lt10 to gt 100 bp average 55 bp
29
D2 Chromatin structure
D2-6. The 30 nm fiber - Steps to make it
30
D2 Chromatin structure
Step 1 form a Nucleosome (???)
DNA Histone octamer (??????) ?Nucleosome core
(????? 146bp) H1 ? Chromatosome (???? 166bp)
linker DNA ? Nucleosome (???) (200 bp of DNA)
Nucleosome (???) is the basic structural subunit
of chromatin, consisting of 200 bp of DNA and an
octamer of histone proteins.
31
D2 Chromatin structure
Step 2 Beads on a string structure
Nucleosome repeat Core linker DNA 200 bp
Histone H1
Nucleosome
Linker DNA lt10 to gt 100 bp average 55 bp
32
The 30 nm fiber
D2 Chromatin structure
30 nm fiber (30nm in diameter) Solenoid (???)
Step 3 30 nm fiber
  • Higher ordered
  • Left-handed helix
  • Six nucloesomes per turn

33
D2 Chromatin structure
Step 4 looped domain structure Highest level of
chromatin organization
30 nm fiber
300 nm
Nuclear matrix (???), protein complex
34
Supplementary 1
Nucleosomes and micrococcal nuclease treatment
35
Supplementary 2
Steps from DNA to chromosome
36
Prokaryotic and eukaryotic chromosome structure
D3 Eukaryotic chromosome (?????)
37
Prokaryotic and eukaryotic chromosome structure
D3-1 Cell cycle (????) (????)
38
D3 Eukaryotic chromosome structure
Cell cycle
Interphase ?? G1 S G2 (G0) M phase
(mitosis ????)
39
D3 Eukaryotic chromosome structure
D3 Eukaryotic Chromosomal Structure
Cell cycle
M phase includes prophase (??), metaphase (??),
anaphase (??), telophase (??)
40
D3 Eukaryotic chromosome structure
Mitotic chromosome
41
D3 Eukaryotic chromosome structure
D3-2 Mitotic chromosome (?????????)
more condensed than chromatin, and most highly
condensed at mitosis
42
D3 Eukaryotic chromosome structure
Telomere
Sister chromatid ??????
Centromere ???
Mitotic chromosome at metaphase
Nuclear matrix ???
Loops of 30nm fiber
Chromatid ????
43
D3 Eukaryotic chromosome structure
The centromere (???)
  1. The region where two chromatids (?????)are joined
  2. The sites of attachment to the mitotic spindle
    (???) via kinetochore (???)
  3. Centromere DNA

Yeast
AT-rich (88bp)
44
D3 Eukaryotic Chromosomal Structure
D3 Eukaryotic chromosome structure
Mitotic chromosome
Mitotic spindle
45
D3 Eukaryotic chromosome structure
Mitotic chromosome - centromere
Yeast centromere
Mammalian cells much longer, flanked by
satellite DNA (??DNA)
46
D3 Eukaryotic chromosome structure
The Telomere (??)
  1. Specialized DNA sequences which form the ends of
    the linear DNA of the eukaryotic chromosome
  2. Contains up to hundreds copies of a short
    repeated sequence (5-TTAGGG-3 in human)
  3. Synthesized by the enzyme telomerase (a
    ribonucleoprotein) independent of normal DNA
    replication.
  4. The telomeric DNA forms a special secondary
    structure to protect the chromosomal ends from
    degradation

47
D3 Eukaryotic Chromosomal Structure
D3 Eukaryotic chromosome structure
Telomere Telomerase
Repeat sequence Tetrahymena- TTGGGG human-
TTAGGG
48
D3 Eukaryotic chromosome structure
Telomere structure
A loop structure forms at the end of chromosomal
DNA
49
Prokaryotic and eukaryotic chromosome structure
D3-3 Interphase chromosomes (????????)
50
Interphase chromosomes chromatin (???)
D3 Eukaryotic chromosome structure
  • Heterochromatin (????)
  • Highly condensed
  • Transcriptionally inactive
  • Can be the repeated satellite DNA close to the
    centromeres, and sometimes a whole chromosome
    (e.g. one X chromosome in mammals)

51
D3 Eukaryotic chromosome structure
  • Euchromatin (????) chromatin other than
    heterochromatin.
  • More diffused and not visible
  • The region where transcription takes place
  • Not homogenous, only a portion (10) euchromatin
    is transcriptionally active where the 30nm fiber
    has been dissociated to beads on a string
    structure and parts of these regions may be
    depleted of nucleosome.

52
D3 Eukaryotic chromosome structure
DNase I hypersensitivity How to map the regions
of transcriptionally active chromaatin ?
53
Euchromatin
D3 Eukaryotic chromosome structure
  • CpG methylation (???) CpG island (CpG ?)
  • Methylation of C-5 in the cytosine base of
    5-CG-3
  • Occurs in mammalian cells
  • Signaling the appropriate level of chromosomal
    packing at the sites of expressed genes
  • CpG methylation is associated with
    transcriptionally inactive regions of chromatin
  • Islands of unmethylated CpG are coincident with
    regions of DNase I hypersensitivity
  • Islands surround the promoters of housekeeping
    genes.
  • Responsible for epigenetic (????)and may also
    to RNA silencing (RNA ??)

54
D3 Eukaryotic chromosome structure
Euchromatin
55
D3 Eukaryotic chromosome structure
Fig. 3. CpG islands and the promoters of
housekeeping genes
56
Supplementary
Transcription nucleosome formation
57
Prokaryotic and eukaryotic chromosome structure
D3-4 Histone variants and modification (?????????)
The major mechanisms for the condensing and
decondensing of chromatin operate directly
through the histone proteins which carry out the
packaging.
58
D3 Eukaryotic chromosome structure
  • Short-term changes in chromosome packing
    modulated by chemical modification of histone
    proteins
  • Actively transcribed chromatin via acetylation
    (???) of lysine residues in the N-terminal
    regions of the core histones.
  • Condensation of chromosomes at mitosis by the
    phosphorylation (???) of histone H1.

59
D3 Eukaryotic chromosome structure
  • Longer term differences in chromatin
    condensation associated with changes due to
    stages in development (??) and different tissue
    types.
  • Utilization of alternative histone variants, H5
    replacing H1 in some very inactive chromatin.

60
Prokaryotic and eukaryotic chromosome structure
D4 Genome Complexity (???????)
61
D4 Genome Complexity
D4-1 Genome (???) genetic complexity
(?????) (????)
62
D4 Genome Complexity
Range of genome size in different phyla
??
??
?? ?
??
63
D4 Genome Complexity
Genome genes (?????????)
Genome all DNA sequences in a cell Genes a
stretch of continuous DNA sequence encoding a
protein or RNA
C-value is the quantity of DNA in the genome (per
haploid set of chromosomes). C-value paradox
(C???) refers to the lack of a correlation
between genome size and genetic complexity
64
D4 Genome Complexity
D4-2 Reassociation kinetics (???????)
  • Genomic DNA extraction ?
  • Sonication or shearing to a uniform size
  • (x 100-1000 bp) ?
  • 3. Thermal Denaturation ?
  • 4. Re-annealing
  • 5. Measure plotting the re-annealing process
    will yield kinetics

65
D4 Genome Complexity
Measuring methods Spectroscopy/UV
absorption hydroxyapatite (?????)chromotography
66
D4 Genome Complexity
D4-3 non-coding sequence
  • DNA sequence that does not code for protein or
    RNA, including
  • Introns (unique sequence) in genes
  • DNA consisting of multiple repeats, can be
    tandemly repeated sequences (??????)(e.g.
    satellite DNA) or interspersed repeats (????)
    (e.g. Alu element) etc.

67
D4 Genome Complexity
D4-4 Unique sequence DNA (????DNA)
  • The slowest to reassociate
  • Corresponds to coding regions of genes occurring
    in one or a few copies/haploid genome
  • All the DNA in E. coli genome has a unique
    sequence. Why E. coli DNA associate fast?

68
D4 Genome Complexity
D4-5 Repetitive DNA (????DNA)
Tandem gene clusters (?????) (1) moderately
repetitive DNA consists of a number of types of
repeated sequence. (2) genes whose products are
required in unusually large quantities, e.g.
there are 10-10000 copies of rDNA encoding 45S
precursor and X100 copies of histone genes.
69
D4 Genome Complexity
  • Dispersed repetitive DNA (??????)
  • Moderately repetitive (x100- x1000 copies)
  • Scattered throughout the genome
  • Human Alu elements 300bp, 300 000 500 000
    copies of 80-90 identity
  • Human L1 element
  • Alu L1 10 of human genome.
  • Functions of these repetitive DNAlargely unknown

70
D4 Genome Complexity
  • Satellite DNA (??DNA, simple sequence)
  • Highly repetitive DNA (gt106).
  • very short (2 to 20-30bp, mini- or micro-), in
    tandem arrays
  • concentrated near the centromeres and forms a
    large part of heterochromatin.
  • as separate band in buoyant density gradient
  • no function found, except a possible role in
    kinetochore binding
  • Minisatellite repeats are the basis of the DNA
    fingerprinting techniques (????), Why?

71
D4 Genome Complexity
Satellite DNA
Mouse genome DNA
  • 30 GC in satellite DNA

CsCl centrifugation
72
D4 Genome Complexity
5 ATAAACTATAAACTATAAACT 3 3
TATTTGATATTTGATATTTGA 5
n
ACAAACT, 1.1x107 bp, 25 genome ATAAACT, 3.6x106
bp, 8 genome ACAAATT, 3.6x106 bp, 8
genome AATATAG, cryptic Satellites comprise more
than 40 of the genome
Drosophila satellite DNA repeat (several million
copies)
73
Supplementary
Genes in Drosophila genome
74
  • Summary
  • Prokaryotic chromosome closed-circular DNA,
    domains/loops, negatively supercoiled, HU H-NS
  • Eukaryotic chromatin Histones (octamer H2A,
    H2B, H3, H4)146bp DNA gt Nucleosomes H1 gt
    chromatosome Linker DNA gt beads on string gt
    30nm fiber gt fiber loop nuclear matrix gt highly
    ordered chromatin gt gt gt chromosome
  • Eukaryotic chromosome structure centromere,
    kinetochore, telomere, hetero- or euchromatin,
    CpG island and methylation
  • Genome complexity noncoding DNA, unique
    sequence, repetitive DNA, satellite DNA

75
Homework (on the CD)
  1. See the animations for DNA topology,
    Topoisomerase, as well as Ribozyme Structure and
    Activity. Answering the questions in applying
    your knowledge is required.
  2. Play the structural tutorial Introduction to the
    DNA structure to better understand DNA structure
  3. Finish all the critical thinking exercise
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