Ch.8 DNA Structure and Analysis, Organization in Chromosomes Flashcards
Chromosomes
Theory of inheritance
Proof that genetic info is passed on with the chromosomes (1940s)
Chromosomes
Genetic info
Chromosomes consist of nucleic acids and proteins. Which one carries the genetic info?
Criteria for Genetic Material
To serve as genetic material, a molecule must be able to: Replicate (by themselves, autonomously) Store information Express information Allow variation by mutation
Central Dogma
DNA -> RNA -> protein
DNA makes RNA (transcription), which makes proteins (translation)
Proof that Genetic info is stored in DNA
In organisms and many viruses, the genetic info is encoded in DNA (some viruses also RNA).
Until 1944: what chemical component makes up genes and is the actual genetic material?
Why the doubt?
Thera are 21 different amino acids but only 4 different nucleotides. (ppl were in favor of proteins being the genetic material - could chemically analyze proteins and DNA)
Can the incredible diversity of life really be based on just 4 nucleotides?
Transformation (Griffith 1929)
Mouse experiment
The traits of being virulent and smooth coating were passed from bacteria to bacteria through transformation
Transformation in vitro (petri dish)
Sia and Dawson transformed bacteria in vitro ans showed that the mouse host plays no role in transformation.
DNA mediates transformation
Avery, MacLeod, McCarty (1944) used specific enzymes to degrade DNA, RNA, or proteins.
DNAse treatment eliminated transformation activity.
Proof that DNA must be the carrier of information.
Hershey and Chase Experiment
Life cycle of a T-even bacteriophage as known in 1952.
Attachment of phage tail fibers to bacterial wall.
What enters the cell and directs phage reproduction?
Phage genetic material (?) is injected into bacterium.
Phage reproduction cycle begins.
Components accumulate; assembly of mature phages occurs.
Cell lysis occurs and new phages released.
Hershey and Chase Experiment 1952
Phosphorus Isotope
Phage T2 added to E.coli in radioactive medium.
Phosphorus isotope.
Phosphorus isotope in DNA
Phosphorus isotope in next generation of virus.
(Were able to radioactively mark DNA or proteins.
Proof that virus consists of DNA and proteins- DNA injected into the bacteria and carries the info.)
Hershey and Chase Experiment 1952
Sulphur Isotope
Phage T2 added to E.coli in radioactive medium.
Sulphur isotope.
Sulphur isotope in protein. (not a component of DNA)
No Sulphur isotope trace in next generation of virus.
(Were able to radioactively mark DNA or proteins.
Proof that virus consists of DNA and proteins- DNA injected into the bacteria and carries the info.)
The genetic material of tobacco mosaic virus RNA
Fraenkel-Conrat (1957) mixed proteins from one virus strain with RNA from another virus strain. When tobacco leaves were infected, the resulting virus offspring was always genotypically and phenotypically identical to the parent strain from which the RNA was obtained.
Concluded - RNA carries information
Eukaryotes Indirect evidence
Close correlation between gametes and diploids in amt of DNA and number of chromosome sets.
No such correlation between gametes and diploids for proteins.
Eukaryotes Direct evidence
Recombinant DNA technology (ex: human insulin gene transferred and expressed into a bacterium) Transgenic animal (ex: Human DNA microinjected into fertilized mouse egg, human B-globin gene, expressed in mouse and transmitted to progeny)
Nucleic acids: Structure of DNA and RNA
Understanding the structure of nucleic acids is cruical to understanding how heredity works on the molecular level.
DNA is usually double stranded with adenine paired with thymine and guanine paired with cytosine.
RNA is usually single stranded and contains uracil in place of thymine.
Nucleic acids structure
Polymers made of nucleotides monomers.
Nucleotides are composed of
1. phosphate group
2. pentose (sugar with 5 carbon atoms): Ribose (in RNA) and 2- in Deoxyribose (in DNA)
3. Cyclic nitrogen-containing base: pyrimidines and purines.
Pyrimidines
Uracil, Cytosine, Thymine
Purines
Adenine, Guanine
Triphosphates
Serve as precursor molecule during nucleic acid synthesis (dNTPs in PCR reactions).
ATP and GTP: Adenosine triphosphate and guanine triphosphate.
-large amt of energy involved in adding/removing terminal phosphate group
4 different deoxyribonucleotides
Deoxythymidine monophosphate, dTMP
Deoxycytidine monophosphate, dCMP
Deoxyadenosine monophosphate, dAMP
Deoxyguanosine monophosphate, dGMP
The phosphate group is attached to the 5’ carbon atom.
The nitrogenous base is attached to the 1’ carbon atom.
(monomers of DNA)
Structure of a polynucleotide chain
Chain single nucleotides together to form polymer.
Phosphodiester linkage (C-O-P-O-C) joins the nucleotides.
The polynucleotide chain is directional.
“Five prime end” upstream
“tree prime end” downstream
The Double Helix
Watson and Crick inferred the DNA double helix model based on 2 discoveries
1) Chargaff’s rule
2) Rosalind Franklin’s x-ray crystallography pictures of DNA molecules.
Chargaff’s Rule (1947)
Measure content of DNA and always find same amounts of G and C and same of A and T - all the %s add up to 100%.
A and T and C and G are present in the DNA molecules in equal amounts.
This is bc of complementary nucleotides pairing in the DNA double helix.
Rosalind Franklin: X-ray Diffraction Pattern of DNA
DNA helical structure.
Bases are stacked perpendicular with a periodicity of 0.34 nm.
The DNA double helix
Right handed double helix, the 2 polynucleotide chains are coiled around each other in a spiral
Covalent bonds
Strong chemical bonds formed by sharing of electrons between atoms.
- In bases and sugars
- In phosphodiester linkages (chaining nucleotides)
Hydrogen bonds
Weak bond between an electronegative atom and a hydrogen atom (electropositive) that is covalently linked to a second electronegative atom.
Strands are bonded with these. Break easily with heat.
Hydrophobic bonds. Van der Waals interactions
The association of nonpolar groups with each other when present in aqueous solutions because of their insolubility in water.
Opposite polarity of the 2 strands
Structure
Bonds
Run in opposite directions of each other.
Sugar phosphate backbone.
Bases in the core.
A-T bonded with 2 H bonds.
C-G bonded with 3 H bonds.
Apply heat and break H bonds, strands separate. Cool down and bonds will form again.
Complementary base-pairing - only possible if bases are arranged in a certain way.
Strands run opposite ways
5’-> 3’
3’ -> 5’
Calculating base content in DNA
If you known 1 you can calculate others. Possible in double stranded DNA bc of complementary base pairing 33% guanine. How much adenine is there? 33% cytosine 34% adenine and thymine 17% adenine 17% thymine
Alternate forms of the double helix
A-DNA
Very rare.
Dehydrated condition, high salt conc.
Right handed
11 bp per turn
Alternate forms of the double helix
B-DNA
Most common
Under physiological conditions (aqueous solution, low salt conc.)
Right handed.
10 bp per turn
Alternate forms of the double helix
Z-DNA
Function unknown
Left handed
12 bp per turn
Supercoiling
Functional DNA molecules in living cells are negatively supercoiled.
Regulated by enzymes with essential roles during DNA replication.
Only in DNA molecules with fixed ends (attached to something on its end)
Negative supercoiling
Rotation left handed; underwound DNA -> DNA of all living organisms is negatively supercoiled (very rare exceptions)
Positive supercoiling
Rotation in the same direction as the double helix (right handed); overwound DNA
Structure of RNA
Ribose Nucleic Acid
Sugar ribose replaces deoxyribose of DNA
Nitrogenous base uracil replaces thymine of DNA.
Most are single stranded
Exception: Animal RNA viruses have double stranded RNA helices.
3 major classes of RNAs
3 classes of cellular RNAs (function during gene expression)
rRNA
mRNA
tRNA
rRNAs
Ribosomal RNAs
Structural components of ribosomes for protein synthesis
mRNAs
Messenger RNAs
Template for protein synthesis
Carry genetic info from gene to ribosome
tRNAs
Transfer RNAs
Carry amino acids for protein synthesis
Chromosome structure in bacteria and viruses
Most viruses and bacteria have a single set of genes stores in a single chromosome, which contains a single molecule of nucleic acid (either DNA or RNA).
Bacteria have additional plasmids (not necessary for survival).
The DNA molecules of bacteria and viruses are organized into negatively supercoiled loops (domains)
Reason: space that is available
-huge amt of DNA has to be packaged into small cell
The E.coli chromosome
a) circular, unfolded chromosome
b) folded chromosomes, actually 40-50 loops (looped around RNA)
c) supercoiled folded chromosome
Partial DNase digestion = partially uncoiled chromosome (nicked DNA)
Partial RNase digestion = partial unfolded chromosome (RNA cleaved)
Folded genome: highly condensed functional state of the bacterial chromosome (necessary to organize large amt of DNA into small cell)
Chromosome structure in Eukaryotes
Much more complex than prokaryotic chromosomes.
Multiple chromosomes, each with a single large DNA molecule and considerable amounts of proteins.
Change appearance during cell cycle.
Interphase: chromosomes are metabolically active: chromosomes nearly invisible (DNA replication).
Metaphase on mitosis and meiosis: chromosomes clearly visible (condensation)
Amt of DNA in Eukaryotic chromsomes
varies considerably.
No correlation between chromosome size and number of base pairs in a chromosome.
Packaging of chromatin in eukaryotic chromosomes
Metaphase chromosomes consist of chromatin fibers (not only DNA molecules)
Chromatin composition
Chromatin: complex of DNA, RNA and protein in the nucleus.
Proteins: histones (basic, positively charged), nonhistone chromosomal proteins (acidic, negatively charged).
5 diff types of histones (H1, H2a, H2b, H3, H4)
Nucleosome structure
Nucleosome core - octamer of histones -2 H2a + 2 H2b + 2 H3 + 2 H4 Complete nucleosome -histone H1 stabilizes the structure Histones in core and DNA wrapped around.
Nucleosomes
Chromatin subunit.
Beads on a string - nucleosome isolated from interphase nuclei.
Nucleosome core - 146 nucleotide pairs Of DNA wrapped as 1 3/4 turn around an Octamer of histones.
Linker DNA varying in length from 8 to 114 nucleotide pairs.
DNA around a scaffold of nonhistone proteins
Giving metaphase protein it’s shape.
Metaphase chromosomes from which histones have been removed. (DNA stretched out.
DNA is organized around a central core of nonhistone protein (=scaffold)
Levels of DNA packaging
2nm double stranded DNA molecule.
11 nm nucleosomes.
30 nm chromatin fiber.
Organization around a central scaffold. (Chromosome shape)
Special features of Eukaryotic Chromosomes
Contain genes.
But also contain nongenic parts: DNA that does not encode information for proteins or specific RNAs.
Much of this nongenic DNA consists of repetitive sequences.
Called junk DNA but they are important in chromosome structure.
Satellite DNA
In junk DNA.
G:C and A:T rich DNA
Repetitive sequences.
Degree of repetition: copy number can vary (10^3 - 10^6).
DNA contains a mixture of unique, moderately repetitive and highly repetitive sequences.
Satellite sequences in human centromeres
Without centromeres, chromosomes would not be able to divide.
Hold the kinetochores (the protein structures the microtubules attach to during metaphase).
Functional centromeres are important (chromosomes without centromeres will be lost during cell division) (microtubules won’t attach).
Yeast: DNA segment 125 bp long.
Multicellular plants and animals: thousands to millions of bp long.
Humans: up to 1.5 million bp long with up to 15,000 copies of a 171 bp long sequence called alpha satellite sequence.
Telomeres
Tip of chromosome arms.
Repetitive sequences in distinct structures at the top of the chromosome arms: telomeres.
Function of telomeres
-protect the ends of linear DNA molecules from DNAses (bacteria DNA is circular so it doesn’t have this prob)
-prevent fusion of chromosomes
-facilitate complete replication of the ends of linear DNA molecules.
Nucleosomes
Chromatin subunit.
“beads on a string” nucleosine substructure isolated from interphase nuclei.
Nucleosome core, 146 nucleotide pairs of DNA wrapped as 1 3/4 turns around an octamer of histones.
Linker DNA, varying in length from 8-114 nucleotide pairs.
DNA around a scaffold on nonhistone protiens
giving metaphase chromosome its shape.
Metaphase chromosomes from which histones have been removed. (DNA stretched out)
DNA is organized around a central core of nonhistone protein (=scaffold).
Levels of DNA packaging
2nm - double stranded DNA molecule
11nm - nucleosome
30 nm - chromatin fiber
organization around a central scaffold - chromosome shape.
Special features of Eukaryotic Chromosomes
(distinguishes them from bacteria chromosomes)
Contain genes.
But also contain nongenic parts: DNA that does not encode information for proteins or specific RNAs.
Much of this nongenic DNA consists of repetitive sequences.
Called junk DNA, but they are important feature in chromosome structure.
Satellite DNA
In junk DNA.
G:C and A:T rich DNA.
Repetitive sequences.
Degree of repetition: copy number (ex: 10^3 - 10^6).
DNA contains a mixture of unique, moderately repetitive and highly repetitive sequences.
Satellite Sequences in Human Centromeres
(Without centromeres, chromosomes would not be able to divide).. Hold kinetochores (the protein structures the microtubules attach to during metaphase). Functional centromeres are important (chromosomes without centromere will be lost during cell division). Yeast: DNA segment 125 bp long Multicellular plants and animals: thousands to millions of bp long. Humans: up to 1.5 million bp long with up to 15,000 copies of a 171 bp long sequence called alpha satellite sequence.
Telomeres
(Tip of chromosome arms)
Repetitive sequences in distinct structure at the tip of the chromosome arms: telomeres.
Function of telomeres
Protect the ends of linear DNA molecules from DNAses (bacterial DNA is circular so it doesnt have this prob).
Prevent fusion of chromosomes
Facilitate complete replication of the ends of linear DNA molecules.
Telomeres protect chromosome ends
Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules.
It has been proposed that the shortening of telomeres is connected to aging (every time a DNA molecule replicates, you lose a tiny bit of DNA molecule on the linear ends.
Telomere structure
Basic repeat units: 5’ T (1-4) A(0-1) G (1-8) 3’.
Normal human somatic cells contain 500 to 3000 TTAGGG repeats.
Gradually shorten with age.
Telomere structure in humans: 3’ overhang, t-loop and sheltering protein complex (stabilizes)
Analytical Techniques
(based on chemical nature) Useful during DNA and RNA investigation. Absorption of UV light. Denaturation and renaturation of nucleic acids. Molecular hybridization. FISH: Fluorescent in situ hybridization. Electrophoresis of nucleic acids.
Absorption of UV lights
Nucleic acids
Absorb UV light strongly at 254-260 nm due to interaction between UV light and ring systems of the bases.
UV light used in localization, isolation, and characterization.
Use of UV critical to isolation of nucleic acids following separation.
Visualization of nucleic acids on agarose gels under UV light.
Denaturation and Renaturation of nucleic acids
DNA can denature due to heat or stress.
Hyperchromic shift (after denaturation):
Increase in UV absorption of heated DNA in solution.
Denaturation used to determine melting temperature (of DNA molecules).
Melting profile of DNA
For 2 molecules with different G-C contents
The molecule with a melting point of 83 degrees has a greater G-C content than the molecule with a melting point of 77 degrees.
Higher G-C content- higher melting point
AT has 2 H bonds
CG has 3 H bonds
More H bonds, the higher the melting point
Molecular hybridization
Denature and renaturation of nucleic acids are the basis for molecular hybridization.
Ex: single strands of nucleic acids combine duplex structures, yet are not from the same source. (strands from different sources renature and form double stranded hybrids).
If DNA is isolated from 2 distinct sources, double stranded hybrids will form.
Can be used to design primers for PCR rxns, and probes to tag DNA sequences.
FISH: Fluorescent in situ hybridization
Uses fluorescent probes to monitor hybridization
Probes are nucleic acids that will hybridize only with specific chromosomal areas.
Mitotic cells fixed to slides and subjected to hybridization
ssDNA is added and hybridization is monitored.
Electrophoresis
Separates DNA and RNA fragments by size.
DNA and RNA have a negative charge.
Molecules migrate to the plus pole when subjected to a current.
Smaller fragments migrate through the gel at faster rates than large fragments.
Agarose gel - porous matrix restricts migration of larger molecules more than it restricts smaller ones.
Agarose gel
PCR
DNA is neg charged molecule.
When a current is applied, DNA travels through the gel towards the positive pole.
The polymer gel works like a sieve.
Smaller (shorter) pieces travel faster than larger (longer) pieces.
Measure quality of DNA extractions.
Visualize results from PCR.
1 well is used for a size standard - mixture of DNA fragments of definite sizes, compare other bands to standard.
Can determine size and see if its the DNA you are looking for - look at particular gene.
