Transcript Organization of DNA • Viral DNA (and in some, RNA)

Organization of DNA
• Viral DNA (and in some, RNA)
– Some circular, some linear
– Some double stranded, some single stranded
– Very small amount, packed very tightly
• Small size is an advantage
• Viruses use host cell enzymes, need few genes
• Bacterial DNA
– Usually single copy of double stranded
– Usually circular
• Eukaryotic DNA: linear, in several pieces
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DNA packaging
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For example, the chromosome of E. coli is 1.2 mm long,
but must fit into a bacterium that is only 0.001 mm long!
http://www.expatica.com/xpat/xpatsite/www/upload_pix/surprised-face.jpg
Protein packaging of DNA
• Four proteins in E. coli
– Make up 10% of total protein of cell
– HU for wrapping; FIS and IHF for bending; HNS for
compaction.
– Same function as histone proteins in eukaryotes.
• Positively charged proteins bind to negatively
charged DNA.
– End result: nucleoid, a region in the cytoplasm rich
in DNA and protein; comparable to a nucleus but
without a membrane.
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DNA of E. coli is supercoiled
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• In addition to being packaged with proteins, the
DNA of E. coli is supercoiled.
– Supercoiling. DNA could be “relaxed” or
supercoiled. In Eubacteria, DNA is “underwound”
(negatively supercoiled);
– Supercoiling carried out by topoisomerases.
• Example: gyrase, that relieves stress during DNA
replication.
• Two types, depending on whether 1 or two DNA
strands are cut (and repaired) in the process.
Supercoiling
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Top left: relaxed DNA
Bottom left: supercoiled.
Bottom: schematic of underwinding
DNA.
Packaging of E. coli DNA
Note arrows: one shows
where the DNA has been
“nicked”, relaxing the
supercoiling. The other
points to a supercoiled
region.
That supercoiling can be
relaxed in ONE PLACE
means that the DNA is
constrained in places.
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The enslaved bacteria
• Mitochondria and chloroplasts thought to have
originated as prokaryotic endosymbionts in
early eukaryotes
– Carry out respiratory functions in membrane
– DNA is circular, ds DNA like in prokaryotes
– Self replicating
– Have their own ribosomes, similar to bacterial
• Organelle DNA discovered from mutations
– Some traits not determined by nuclear genes
– Inheritance via mother; ovum has all the cytoplasm
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Integration of organelles is thorough
• Mitochondria:
http://cellbio.utmb.edu/cellbio/mitoch2.htm
– Replication requires nuclear genes
– Polymerases, initiation factors, respiratory proteins
are multi-subunit proteins
• Several of the subunits for each are nuclear,
others are mitochondrial
• Chloroplasts
– Multi-subunit enzymes jointly encoded
– Genes for RuBP carboxylase divided between
nucleus and chloroplast
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Polytene chromosomes
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Occur in the salivary glands of various flies during
development.
Condensed areas of DNA line up, produce darkly staining
bands.
Useful for mapping genes: banding patterns are unique, and in
situ hybridization can be used to localize genes on DNA
DNA packaging in eukaryotes
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• Largest human chromosome is made of DNA which is
82 mm long (over 3 inches)
• During metaphase, DNA is further compacted to about
10 µm long.
– Equivalent to winding 25 miles of spaghetti into a 16
foot canoe.
• DNA has to be well packaged to fit into the cell, to be
compacted even more during mitosis
– still has to be accessible during interphase for use!
• Chromatin: grainy appearing mixture of DNA and
proteins in the nucelus
Nucleosomes: unit of packaging of
eukaryotic DNA
DNA wrapped around
histone proteins:
TWO each of the
proteins H2A, H2B,
H3, and H4.
Additionally, H1 on
outside helps hold
DNA to structure.
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About histones and arrangement
• Histones
– positively charged, to attach well to DNA
– conserved, very little difference among organisms
• How arrangement was determined
– DNA collected, treated briefly with nuclease to
see how much DNA is protected by proteins
– Remove proteins, separate DNA pieces by size
on gel
– 200 bp pieces of DNA produced
– treat more with nuclease, repeat analysis
– get 145 bp DNA pieces
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Structure deduced
• the 145 bp of DNA are
wrapped around the
histone octet which is
the core particle.
• 200 bp includes region
covered by H1 which
covers DNA as it enters,
exits nucleosome.
• the rest of the DNA is
linker DNA between.
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Nucleosomes are wound up
Figure shows how
“beads on a string” are
further wound up to
produce a solenoid, the
structure of chromatin.
During mitosis, this
solenoid itself coils
further to make
chromatids.
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Organization of DNA
• Does DNA packaging create problems?
– DNA wrapped tightly around histones
– DNA must be accessible for replication,
transcription
• Modification of histones changes packing with DNA
– Acetylation: acetylases added to histones.
– Phosphorylation: phosphate groups added by
kinases
– These groups decrease positive net positive
charges, allow DNA freedom.
– Negative supercoiling helps too.
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Differences in DNA
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• Heterochromatin vs. Euchromatin
– Heterochromatin is DNA which tends to be highly
compacted and dark staining.
– Euchromatin is not so compacted or dark.
– The number of genes in heterochromatin is
generally small relative to euchromatin.
• Heterochromatin lacks genes or they are inactive
• Much heterochromatin is found in certain structural
parts of the chromosomes: centromeres and
telomeres. Also, much of Y chromosome.
– Move euchromatin to an area next to
heterochromatin and it becomes heterochromatin:
position effect.
Chromosome structure
Arm
http://www.med.uiuc.edu/m1/genetics/images/webun1/Chromosome.gif
medic.med.uth.tmc.edu/.../ cellbio/hist-01.htm
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Composition of DNA:
% G+C
There is always equal #s of A and T, and G and C, but the
percentage of G+C pairs and A+T pairs can be different
among different organisms.
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Measuring % G+C
hyperchromic shift
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As DNA “melts”, becomes SS, absorbs more UV at 260 nm.
Because G-C pairs have 3 H-bonds instead of two, DNA with
more G+C is more stable, melts at higher temperature (blue).
Satellite DNA
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• In prokaryotes, the %G+C base pairs is pretty
much averaged out over the entire DNA; not so
with eukaryotes.
• Density gradient ultracentrifugation can also be
used to determine %G+C.
– G+C pairs are denser than A+T, migrate to a lower
location (greater density) in the gradient.
• Fragmented eukaryotic DNA showed something
odd…
Satellite DNA
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When the DNA was
analyzed, a portion has
a lower %G+C than
the rest of the DNA,
producing a “satellite
band”. How could a
portion of DNA have a
different composition
than the rest?
Repeated sequences
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• If a section of DNA with a %G+C composition
different from the rest of the DNA is repeated
many times, DNA fragments from these regions
of DNA would behave differently during the
centrifugation.
Study of the Composition of DNA using
DNA renaturation kinetics
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• Break DNA into random fragments.
• Denature with heat (melt).
• Cool, allow strands to find their complements
and go from ss to ds again (anneal).
• Follow entire process using UV light absorption
at 260 nm
– as DNA goes from ss to ds, Abs decreases.
Renaturation kinetics
• Kinetics: study of the rate of change.
• Major Point #1: the more copies of the
complementary strands there are, the less time they
will take to
find each other
– the more DNA,
the faster the process.
In this fig., 2 different amounts of
DNA from the SAME organism.
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Renaturation kinetics-2
•Major Point #2:
Given equal amounts
(same mass) of DNA,
the bigger the total genome
of the organism, the slower
the renaturation.
–If the genome is bigger, and the amounts of DNA used
in the experiment are the same, the organism with the
bigger genome will have fewer copies of the
complementary fragments, so annealing will take longer
(see point #1).
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Understanding genome size
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Imagine you have 20 playing
cards. In one instance, you have
these 5 cards, another 5 cards
exactly the same, and 2 more sets
of the Ace thru 10 but of
diamonds. <Deck 1>
In the second instance, you have
ace thru 5 of hearts and also of
diamonds. <Deck 2>
In which case will you match up pairs of hearts and diamonds
most quickly? The Deck 1 gets matched up quicker.
http://www.skydiveelsinore.com/calendar/images/playing-cards-spread.jpg
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Cot curves: Studying renaturation of DNA
The amount of DNA affects the rate at which DNA fragments
renature. To avoid the problem of comparing samples with
different amounts of DNA, the change in ss DNA is graphed
vs.the initial DNA concentration (Co) x the time (t): Cot
Y-axis is the fraction or
percent of the DNA that is
ss (experiment starts by
denaturing the DNA).
X-axis is Cot which is a
Log scale.
www.cas.muohio.edu/.../gene2000/ lect7/fig9p8c.jpg
Satellite DNA and Cot curves
When human DNA
was analyzed this
way, this was the
result:
Remember the card deck experiment: when there is
only one of each card in the deck, they take longer to
match up. So DNA that anneals quickly must be in
multiple copies…
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Cot curves and satellite DNA
Categories variable among
different organisms.
Highly repetitive DNA, many complements, find each other
quickly. Single copy (unique sequence) much slower.
http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/eukarychrom/cot2.gif
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Types of DNA
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• Unique, single copy: typically 30-75% of DNA in most
eukaryotes.
• Highly repetitive DNA: 5-45 % of DNA depending on
species. In humans:
– ALU family: contains Alu I site. 300 bp long,
appears 500,000 times, dispersed. 5% of DNA.
• SINEs = short intersperesed elements
• transposable
– Alpha satellite DNA: tandem repeats of 170 bp
occur 5,000-15,000 times; make up part of
centromere. 6%
– L1 family (in humans), example of LINEs
• Long interspersed elements
• transposable
Middle or moderately repetitive DNA
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• Moderately repetitive DNA:
– Tandem or interspersed repeats
– VNTRs, good for DNA fingerprinting
• Variable number tandem repeats
• 15 – 100 bp long, between or within genes
– Dinucleotide repeats (CA)N, also good for forensic
work
– in maize and yeasts: transposons in large numbers.
– genes for rRNA, tRNA, ribosomal proteins, histones
All your DNA codes for proteins?
Sorry, not close
• Only 4% codes for proteins, in 30,000 genes
• 96% of DNA includes
– Introns, “junk” DNA within and around genes.
– Genes coding for rRNA and tRNA
– Junk DNA called repetitive sequences
– Pseudogenes; have sequences that look like
genes but are never expressed, don’t work.
• We are related to everything else
– Our genes look like those from chimpanzees,
bacteria.
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