| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
Outline:
A. Organization of Genes on Eukaryotic Chromosomes
Higher eukaryotic DNA contains much DNA that does not encode Protein or RNA, and thus does not comprise Genes [Brown, Fig 1.5, Fig 1.7, ]
Function of this DNA is unknown and may be nonfunctional DNA that is "going along for the ride", and is thus sometimes called Junk DNA or Selfish DNA.
Consistent with this observation of nonfunctional DNA, amount of DNA (haploid content is called the C-value) does not always correlate with complexity of the organism, e.g. amphibians and some plants, e.g. lilies, have much more DNA than humans; this non-correlation is the C-value paradox [Brown, Table 1.1]
1. Classes of Eukaryotic DNA [Brown, Chapter 6.3]
Solitary genes versus Gene Families, which mostly arise by tandem duplication mutational events [Brown, pages 120-124]
Pseudogenes - nonfunctional, mutated tandem duplications
of functional genes
[Brown, page 124]
Tandemly repeated arrays of
genes, to obtain a very high level of gene expression in a short
period of time - tRNA and rRNA structural genes, ribosomal protein
genes, histone genes
[Brown, page 122]
2. Repetitious DNA [Brown, Chapter 6.3]
Discovered through reannealing or reassociation experiments
Reannealing, reassociation,
renaturation, hybridization - all mean the same thing:
complementary ssDNA strands finding each other and forming H-bonds
and dsDNA
Reassociation experiments are
usually called cot curve experiments:
When one permits a solution of complex species of DNA strands
to reanneal, the reaction is a second order kinetics reaction
(two strands have to find each other and reanneal), and the critical
parameter is the product of the DNA concentration Co
and the time t of annealing.
Complexity of DNA is a qualitative measure of the relative amount of repetitive DNA present in a DNA fragment population, e.g. that isolated from a given organism. Thus, the complexity of bacterial DNA is low, whereas that from Metazoans is high. The complexity of the DNA from small viruses or from plasmids is very low: only 'uniques' (unique sequences) and low MW DNA.
Note: Although eukaryotic chromosomes contain a single DNA molecule, this molecule is so large that it is nearly always fragmented into many fragments upon isolation from cells. So one always deals with populations of many DNA fragments. The size of these fragments depends on the severity of the isolation procedure, and varies usually from a few hundred kb to up to a megabase (1,000,000 bp; mb) of DNA.
Three types of DNA, as classified by repetitive structure:
a. Highly repetitive DNA - short sequences (2-25 bp) repeated many times in tandem ... 10-15% of much eukaryotic DNA; amount is species dependent - often found in centromeric regions of chromosomes - can often isolate such as satellite bands (different %(G+C) from main DNA) in CsCl gradients [Brown, Tech Notes 6.1]
Microsatellite DNA - tandem repeat size 2 - 7 bp ...
particularly useful in Genetic Profiling and as markers in Physical
Mapping and Genome Sequencing.
Minisatellite DNA - tandem repeat size of about 25 bp ...
less evenly distributed throughout genomes than microsatellite
DNA
Note: CsCl gradients will be discussed under DNA replication
Examples: Brown, Fig 6.16, Fig 6.17
The number of tandem repeats in STRs (Short Tandem Repeats) often varies from one human to another, and hence has application to forensic science. Using these, one can generate a Genetic Profile of any given human being [Brown, Fig 6.17]
b. Moderately repetitive DNA - composed of tandem repeats of moderately long sequences (100-400 bp, sometimes up to 1000 bp)
Again, variable numbers of these repeats at a given DNA locus are found from individual to individual; hence these are often called VNTRs (Variable Number of Tandem Repeats)
Mobile DNA elements are often found in this type of DNA
... [Brown, Section 6.3.2]
Other Mobile DNA elements, e.g. Retroelements, are more properly
classified as Gene Family DNA.
B. Eukaryotic Chromosome
Structure - Chromatin
... [Brown, Sections
6.1.1 and 8.1]
Chromatin ... the DNA-protein complex present in Interphase cells
EuChromatin ... all chromatin except HeteroChromatin ... chromatin regions that are relatively open, less condensed ... where gene expression occurs
HeteroChromatin ... genome or chromatin regions that are ALWAYS highly condensed, ala Mitotic Chroms ... little gene expression in these regions
Heterochromatin is of two types:
Constitutive HeteroChromatin: Specific genome regions, often containing short repeated sequences
Facultative HeteroChromatin: Entire chromosomes that are thereby transcriptionally inactivated
Key example: Inactive X Chromosome in females
Only one of the two X chromosomes is active in gene expression
C. Structure of Chromatin:
... [Brown, Section 6.1.1]
1) Components
DNA RNA ... nascent transcripts ... 10% amt of DNA
Proteins:
Histones ... small, basic proteins that bind DNA in seq-indep manner
5 classes: H1 H2A H2B H3 H4 ... all small (11-15 kd) except H1...
H3 and H4: highly conserved seq/struc in bio kingdom ... small
H1: variable bet tissues/organisms ... larger (23 kd)... several types: Linker Histones
NonHistones ... various sized proteins ... many
Acidic Proteins
also many DNA function proteins: RNA polymerases, transcription
factors, ...
2) Three levels of Organization - Structure involving Histones:
(1.) 10 nm Nucleosomes: [Brown,
Section 6.1.1]
When Chromatin is isolated in low salt, see "beads on a string":
approximately 200 bp DNA wrapped
around 8 (octamer) histone proteins -
bead-like 10 nm particles, equal
mass of DNA and protein ... PR = 6
Histones: 2 each of H2A, H2B,
H3, H4 - Core Histones
Linker Histone H1:
1 per Nucleosome and on outside of Core Nucleosome
a. Expt: ... [Brown, Fig 6.1]
Digest DNA with Micrococcal Nuclease (DNA endonuclease) ...
digests DNA between Nucleosomes,
releases 90% of DNA in ~200 bp beads
b. Variability in amt of DNA per Nucleosome: 155 to 260
bp
due to variable length of DNA
between Core Nucleosome ... beads on string
DNA in Core Nucleosome:
145 bp ... DNA in "linker": 10-115 bp
c. Histone Octamer: cylinder - 6 nm ht x 10 nm diam (31
nm circum)
(H3 -H4)2 form 'plate', with one (H2A-H2B) above and one below .. [Brown, Fig 8.5]
d. DNA: have 145 bp => for B-DNA, 145 x 0.34 nm = 49.3
nm
This is nearly twice the circumference of the Histone Octamer,
suggesting that
the DNA wraps twice around Histone Octamer ...
Actually have: 1.75 turns of DNA in Left-handed Helix, or Negative,
SuperHelix:
Also: The DNA nearly fills the 6 nm Height
of the Histone Octamer:
2 x 20Å = 2 x 2 nm
= 4 nm, nearly fills the 6 nm Height
e. X-ray diffraction structure: ... [Brown, Fig 8.5]
Drawing: Histones: alpha/alpha structure, long central alpha helix, ala Calmodulin
H3-H4 more or less pair together and H2A-H2B more or less pair together
DNA phosphate neg charge interacts with Arg and Lys residues ... V-V, Figure 33-7
Each Histone has N-terminal
Tails that extend out into medium ...
Acetylation state of Lysine residues in these tails is
important in role of Nucleosomes in regulation of Gene Expression:
acetylation is present in nucleosomes in regions of gene expression
f. DNase I (panc DNase) digest of DNA - in agreement:
10 bp repeat of DNase sensitive sites --> double helix sits on outside of Histone surface
Some sites more sensitive than others --> DNA stretched to some extent ... curvature varies some
Repeats confirmed and extended by Hydroxyl radical footprinting
g. Repeats also show Symmetry of DNA entering and leaving Nucleosome
Histone H1: "lock" DNA on Histone Core
at entry-exit point ... [Brown, Fig 6.2]
h. Supercoil Density in Nucleosomes:
Expt: use test DNA ... SV40 DNA: 5200 bp, genome of small animal virus
purify nucleosome-containing SV40 DNA from cultured cells
Relax supercoils via Topoisomerase
I or II ...
{Any remaining supercoils due
to Nucleosomes constraining them, preventing them from being relaxed by Topoisomerase I or II}
Now remove histones ...
Measure Supercoils now present
...
Find 1 supercoil per Nucleosome ... Linking Number Paradox
different from -1.75 neg turns per nucleosome
Why? distortion in Watson-Crick B-form helix of DNA wrapped
around Histone Core ...
~ 1 W-C turn removed from DNA per Nucleosome ...
Note: This fits well with the dimensions involved:
(2.) 30 nm fiber: PR ~ 40 ... [Brown, Fig 6.3]
coiling of Nucleosomes into
Helical Array ... present in Higher Salt
additional proteins required ... present in Interphase and Mitotic
Chromosomes
a. Requires presence of Histone H1
b. Structure: Right-handed "super-super helix",
6 Nucleosomes per turn, in
"solenoid" structure ... stabilized by Histone H1 solenoid
down middle ...
(3.) Fiber Packaging: PR ~ 1000 in Euchromatin; PR ~ 10000 in Mitotic Chroms
Little information currently available ... tough problem ... many artifacts ...
"Coil upon coil upon coil", until complete chromosome structure is obtained ...
[Brown, Fig 6.4]
D. Chromatin Structure in Absence of Histones:
Remove Histones by treatment with mild detergent, e.g. Li diiodosalicylate (LIS) ... then see:
Supercoiled Domains similar to bacterial nucleoid ...40-100
kb in size ... [Brown, Fig 8.3
Nuclear Matrix is present in Interphase Cells
Structure on interior of Nuclear Membrane in Nuclei, to which DNA may well be attached
The "locks" separating
the Supercoiled Domains may be elements of the Nuclear
Matrix ...
MAR - Matrix Attachment Regions ... [Brown, Fig 8.3]
SAR - Scaffold Association Regions ... same thing: MAR = SAR
DNA sites attached to the Nuclear Matrix
Are there specific DNA sequences in these DNA MAR?
Some evidence for this ... Experimental
approach:
1. Isolate nuclei, extract histones; restriction digest the DNA; examine fragments on gels, compare those remaining inside nuclei (MAR fragments) with those that diffuse out of the nuclei (free DNA)
This experiment ASSAYS DNA r.fragments that remain bound to Nuclear Matrix (in vivo)
2. Isolate nuclei, extract histones; digest ALL DNA with pancreatic DNase; add back restriction digested total DNA; analyze DNA that binds on gels: MAR fragments
This experiment ASSAYS DNA r.fragments that can bind to Nuclear Matrix (in vitro)
Mitotic DNA ... depleted of Histones ... still have central protein Scaffold from which DNA loops 30-100 kb in size emanate ... [Brown, Fig 8.3]
These loops are likely to be the Supercoiled Domains
Scaffold has Structure similar to that of Sister Chromatids (daughter chromosomes, attached together at Centromeres during Mitosis) Fibers ... often join the two 'sister chromatid' structures ...
The MAR sites for Interphase chroms may be the same sites that bind Mitotic DNA to the Scaffold in Mitotic cells
No good Consensus Sequence between different MAR DNA sequences
Organization of DNA on the Scaffold: largely unknown in detail
Helical structure on top of helical structure ...
DNA per chromosome is probably a single DNA molecule
Shown by electron microscopy for one Drosophila chromosome ...
Structure and positions of genes along a Chromosome reproducible
Banding patterns: G bands (Giema), Q bands (Quinacrin), Hoescht bands ... [Brown, Table 6.2, Fig 6.4, Research Brief 3.1]
E. Assembly of Nucleosomes
Assembly occurs during DNA Replication / Chromatin duplication
Nucleosome and other structure must be flexible enough to permit DNA function: Replication and Transcription
Replication occurs at forks where the 2 DNA strands separate
Many proteins are involved, forming large Protein Complex at the forks
Nucleosomes must be displaced for this to occur ... they must then reassemble
Assembly: Nucleosomes quickly form on Daughter DNA behind forks
How does this occur ??? ...
Prevalent ideas:
1. Preformed Octamer hops on ... unlikely
CsCl gradients: Octamer is not conserved ...
Evidence suggests the Dimers (H3-H4,
H2A-H2B) are conserved.
2. Octamer assembles on DNA ... likely
Conserved Histone units are likely to be Dimers: H3-H4 and H2A-H2B
Assembly occurs rapidly, promoted by Chaperonins: Protein that facilitate folding of Proteins and formation of Protein complexes
Chaperonin candidates:
Nucleoplasmin bind H2A-H2B
N1 binds H3-H4
Disassembly: little known ... part of Histone Core may remain associated with DNA, eg an H3-H4 dimer, or perhaps the (H3-H4)2 tetramer
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith