Ch.8 DNA Structure and Analysis, Organization in Chromosomes

Question 1

Chromosomes

Theory of inheritance

Answer 1

Proof that genetic info is passed on with the chromosomes (1940s)

Question 2

Chromosomes

Genetic info

Answer 2

Chromosomes consist of nucleic acids and proteins. Which one carries the genetic info?

Question 3

Criteria for Genetic Material

Answer 3

To serve as genetic material, a molecule must be able to:
Replicate (by themselves, autonomously) 
Store information
Express information
Allow variation by mutation

Question 4

Central Dogma

Answer 4

DNA -> RNA -> protein

DNA makes RNA (transcription), which makes proteins (translation)

Question 5

Proof that Genetic info is stored in DNA

Answer 5

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?

Question 6

Transformation (Griffith 1929)

Mouse experiment

Answer 6

The traits of being virulent and smooth coating were passed from bacteria to bacteria through transformation

Question 7

Transformation in vitro (petri dish)

Answer 7

Sia and Dawson transformed bacteria in vitro ans showed that the mouse host plays no role in transformation.

Question 8

DNA mediates transformation

Answer 8

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.

Question 9

Hershey and Chase Experiment

Answer 9

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.

Question 10

Hershey and Chase Experiment 1952

Phosphorus Isotope

Answer 10

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.)

Question 11

Hershey and Chase Experiment 1952

Sulphur Isotope

Answer 11

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.)

Question 12

The genetic material of tobacco mosaic virus RNA

Answer 12

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

Question 13

Eukaryotes Indirect evidence

Answer 13

Close correlation between gametes and diploids in amt of DNA and number of chromosome sets.
No such correlation between gametes and diploids for proteins.

Question 14

Eukaryotes Direct evidence

Answer 14

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)

Question 15

Nucleic acids: Structure of DNA and RNA

Answer 15

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.

Question 16

Nucleic acids structure

Answer 16

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.

Question 17

Pyrimidines

Answer 17

Uracil, Cytosine, Thymine

Question 18

Purines

Answer 18

Adenine, Guanine

Question 19

Triphosphates

Answer 19

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

Question 20

4 different deoxyribonucleotides

Answer 20

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)

Question 21

Structure of a polynucleotide chain

Answer 21

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

Question 22

The Double Helix

Answer 22

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.

Question 23

Chargaff’s Rule (1947)

Answer 23

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.

Question 24

Rosalind Franklin: X-ray Diffraction Pattern of DNA

Answer 24

DNA helical structure.

Bases are stacked perpendicular with a periodicity of 0.34 nm.

Question 25

The DNA double helix

Answer 25

Right handed double helix, the 2 polynucleotide chains are coiled around each other in a spiral

Question 26

Covalent bonds

Answer 26

Strong chemical bonds formed by sharing of electrons between atoms. 1. In bases and sugars 2. In phosphodiester linkages (chaining nucleotides)

Question 27

Hydrogen bonds

Answer 27

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.

Question 28

Hydrophobic bonds. Van der Waals interactions

Answer 28

The association of nonpolar groups with each other when present in aqueous solutions because of their insolubility in water.

Question 29

Opposite polarity of the 2 strands Structure Bonds

Answer 29

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'

Question 30

Calculating base content in DNA

Answer 30

``` 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 ```

Question 31

Alternate forms of the double helix | A-DNA

Answer 31

Very rare. Dehydrated condition, high salt conc. Right handed 11 bp per turn

Question 32

Alternate forms of the double helix | B-DNA

Answer 32

Most common Under physiological conditions (aqueous solution, low salt conc.) Right handed. 10 bp per turn

Question 33

Alternate forms of the double helix | Z-DNA

Answer 33

Function unknown Left handed 12 bp per turn

Question 34

Supercoiling

Answer 34

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)

Question 35

Negative supercoiling

Answer 35

Rotation left handed; underwound DNA -> DNA of all living organisms is negatively supercoiled (very rare exceptions)

Question 36

Positive supercoiling

Answer 36

Rotation in the same direction as the double helix (right handed); overwound DNA

Question 37

Structure of RNA

Answer 37

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.

Question 38

3 major classes of RNAs

Answer 38

3 classes of cellular RNAs (function during gene expression) rRNA mRNA tRNA

Question 39

rRNAs

Answer 39

Ribosomal RNAs | Structural components of ribosomes for protein synthesis

Question 40

mRNAs

Answer 40

Messenger RNAs Template for protein synthesis Carry genetic info from gene to ribosome

Question 41

tRNAs

Answer 41

Transfer RNAs | Carry amino acids for protein synthesis

Question 42

Chromosome structure in bacteria and viruses

Answer 42

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

Question 43

The E.coli chromosome

Answer 43

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)

Question 44

Chromosome structure in Eukaryotes

Answer 44

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)

Question 45

Amt of DNA in Eukaryotic chromsomes

Answer 45

varies considerably. | No correlation between chromosome size and number of base pairs in a chromosome.

Question 46

Packaging of chromatin in eukaryotic chromosomes

Answer 46

Metaphase chromosomes consist of chromatin fibers (not only DNA molecules)

Question 47

Chromatin composition

Answer 47

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)

Question 48

Nucleosome structure

Answer 48

``` 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. ```

Question 49

Nucleosomes

Answer 49

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.

Question 50

DNA around a scaffold of nonhistone proteins

Answer 50

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)

Question 51

Levels of DNA packaging

Answer 51

2nm double stranded DNA molecule. 11 nm nucleosomes. 30 nm chromatin fiber. Organization around a central scaffold. (Chromosome shape)

Question 52

Special features of Eukaryotic Chromosomes

Answer 52

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.

Question 53

Satellite DNA

Answer 53

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.

Question 54

Satellite sequences in human centromeres

Answer 54

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.

Question 55

Telomeres

Answer 55

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.

Question 56

Nucleosomes

Answer 56

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.

Question 57

DNA around a scaffold on nonhistone protiens

Answer 57

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).

Question 58

Levels of DNA packaging

Answer 58

2nm - double stranded DNA molecule 11nm - nucleosome 30 nm - chromatin fiber organization around a central scaffold - chromosome shape.

Question 59

Special features of Eukaryotic Chromosomes

Answer 59

(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.

Question 60

Satellite DNA

Answer 60

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.

Question 61

Satellite Sequences in Human Centromeres

Answer 61

``` (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. ```

Question 62

Telomeres

Answer 62

(Tip of chromosome arms) | Repetitive sequences in distinct structure at the tip of the chromosome arms: telomeres.

Question 63

Function of telomeres

Answer 63

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.

Question 64

Telomeres protect chromosome ends

Answer 64

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.

Question 65

Telomere structure

Answer 65

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)

Question 66

Analytical Techniques

Answer 66

``` (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. ```

Question 67

Absorption of UV lights

Answer 67

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.

Question 68

Denaturation and Renaturation of nucleic acids

Answer 68

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).

Question 69

Melting profile of DNA

Answer 69

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

Question 70

Molecular hybridization

Answer 70

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.

Question 71

FISH: Fluorescent in situ hybridization

Answer 71

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.

Question 72

Electrophoresis

Answer 72

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.

Question 73

Agarose gel | PCR

Answer 73

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.