Lec 18 Flashcards
What is the first way to analyze DNA?
Absorption of Nucleic Acid
- Strong absorption at 260nm (where it minimally absorbs)
- UV interacts with rings of bases
What is the second way to analyze DNA
Gradient Centrifugation (put stuff in tube in spin it, big things to the bottom)
1) Velocity Centrifugation
- measures speed of centrifugation
- measured svedberg units (S)
- in general, greater mass results in greater speed, but also depends on shape
2) Density Gradient Centrifugation (CsCl gradient)
- Also called quilibrium centrifugation because molecules migrate until they reach a point of neutral density (called isopycnic point)
- GC-rich DNA is more dense than AT-rich DNA
- The %GC is directly proportional to the buoyant density of the DNA
isopycnic point
in the density gradient centrifugation, the isopycnic point is when the molecules migrate until they reach a point of neutral density
Gel electrophoresis
- Used to separate DNA fragments
- Fragments migrate from negative to positive
- smaller pieces move further than larger pieces
- Migration distance is inversely proportional to the log (DNA fragment size)
What does it mean when you denature DNA?
means breaking the hydrogen bonds and causing the two stand to sepearate
Denaturation Studies
- strand separation (heat or chemical induced)
- Hyperchromic effect
- observe melting profile
- Tm is the temperature at which 50% of the DNA is denatured
- The Tm is the inflection point on the graph
- High GC content results in high Tm
- Tm= MIDPOINT OF THERMAL DENATURATION
Hyperchromic effect
an increase in UV absorption due to denaturation
Relationship between Tm and %GC
Tm=69+(.41)(%GC)
Cot1/2
point at which 1/2 of the DNA is dsDNA
Complexity
length of all unique fragments laid end to end (in bp)
Classification of repetitive DNA (3 classes)
Highly repetitive= more than 100,000 copies usually short sequences
Moderately Repetitive= 10-100,000 copies
Unique= Single Copy
Chromatin
the complex of DNA, chromosomal proteins and RNA within the nucleus
Euchromatin
lighter staining parts of the chromosome during interphase
- actively transcribed genes
- condenses and relaxes (stretches out)
- condenses and decondenses in cell cycle
- most of transcribed region is in euchromatin
Heterochromatin
darker staining parts of the chromosome
- fewer genes
- remains condensed in interphase
- includes regions in the centromeres and telomeres
- generally not transcribed
- usually not involved in crossing over
- replicates late in S PHASE
Constitutive heterochromatin
always heterochromatin
Facultative hetrochromatin
may be euchromatic sometimes
EX: X chromosomes that is Barr Body
Function of Unique DNA
- thought to contain mostly structural and regulatory genes and is usually transcribed.
- Codes for most proteins and enzymes in organism.
- Found in euchromatic regions of chromosome. Comprises 30%-80% of eukaryotic genome
Function of Moderately repetitive DNA
is found in euchromatic regions of chromosome and code for some ribosomal RNAs and other genes where lots of gene product is needed quickly.
May also pay a role in chromosome structure and contain sites where proteins can bind to DNA for gene.
5-80% of genome.
Includes transposable elements
Function of Highly repetitive DNA
is primarily found in heterochromatic regions of the chromosome. Rarely transcribed and function is not known
TMV- Tobacco Mosaic Virus
?
Histones
are basic proteins and have lots of positively charged amino acids which allow them to bind electrostatic ally to negatively charged phosphates on DNA
-are highly conserved especially H3 and H4
H2A and H2B
?
H1
see tissue to tissue difference
Nucleosome
core histone+ 145-147 bp DNA
Chromatosome
nucleosome + H1 histone
Core Histones
2 each of H2A, H2B, H3, and H4
How was the Chromatin Structure Deduced?
deduced by using micrococcal nuclease digestion for varying lengths of time and analyzing the fragments produced by each.
What chromatosomes in a chromatin fiber separated by?
linker DNA
Nuclear Scaffolds
dark central core of histones in mitotic chromosomes seen when histones are removed (use high salt concentration to removed histones)
-anchor the series of 30 nm loops. Each loop has between 20,000-100,000 bp of DNA
SARs (scaffold attachment regions)
sites between transcription regions where scaffolds can bind DNA. Proteins that bind here may be important for getting the proper DNA conformation for expression
Topoisomerase II
is one of the proteins in the scaffold. It can manipulate coiling of DNA by cutting the backbone. The amount of coiling is important in DNA expression.
Topoisomerases
allow supercoiling to occur
Supercoiling
when DNA coils back on itself when it is overwound or underwound
Human Banding Patterns
produced by treating human chromosomes with enzyme trypsin followed by stain glemsa
Endopolyploidy
several rounds of DNA replication without separation of replicate chromosomes
Bands
are characteristic for strain of organism and can be used to identify particular chromosomes
Puffs and Balbiani rings
are areas where the DNA is loosely coiled so that transcription can occur
Centromeres
- must attach to kinetochore which bind to spindle fibers so reasonable to have common sequence to allow same function
- most of centromere is heterochromatic with short DNA sequences repeated many times
- no specific sequence found in all centromeres
- Do see a different histone (CenH3) that replaces H3 in centromeres of humans, flies, and arabiopsis that seems to bring about a change in chromatin structure to allow formation of kinetochore and binding of spindle fibers
Yeast model for centromeres
contain common central region (82-89bp that is more than 90% AT) surrounded on both sides by conserved regions 11-14 bp long
Telomeres
- provide stability for ends of chromosomes so that chromosomes are not degraded by exonuclease
- prevent chromosomes from joining with each other at the ends due to ligase activity
- provide proper replication of end of chromosome
- although sequences vary from species to species, they are oriented GC pairs toward end of chromosome
What is the human telomeric sequence?
5’- TTAGGG -3’
repeated 300-500 times
T-loop
?
Barbara McClintock
transposable genetic elements “jumping genes”
- piece of chromosome can move from one site to another site (can move to a different chromosome)
- Can alter phenotype when they move (either by disrupting gene or disrupting regulatory area)
The Ac-Ds System in Maize
discovered by Barbara McClintock
Ac- (activator)- required for the Ds element to Move
Ds- (dissociation)- does not contain gene for transposition so it must be directed to move by Ac
Replicative Transposition
uses transposes to move a copy
Nonreplicative Transposition
uses transposes to move original, but doesn’t repair DNA
Conservative Transposition
moves original and repairs DNA
Retrotransposons
use reverse transcriptase to create DNA from element’s RNA
Common Sequence Elements
- sequence is flanked by inverted repeats
- after insertion, the insert sequence is flanked by direct repeats
Ti Plasmid
the Ti plasmid is a tumor inducing plasmid from Agrobacterium tumefaciens. It causes crown gall disease in the normal infection process
-the Ti plasmid inserts into the plant chromosome to cause the disease
Do transposons serve a purpose?
- about 45% of human genome appears to be remnants of transposons (remenant=dont move)
- About 50% of spontaneous mutations in Drosophila are due to transposons
- Transposons have the potential to move regulatory sequences to new places which could affect gene expression
