Chapter 9

Friedrich Miescher

the first chemist to isolate DNA

studied pus samples obtained from hospital bandages at Tubingen University (Germany)

separated the nuclei from the cytoplasm of white blood cells, isolated “nuclein” in 1869

nuclein

the substance isolated from white blood cells by Friedrich Miescher

contained large amounts of phosphorous, but no sulfur

based on lack of sulfur, concluded: not a protein

known today as DNA (deoxyribonucleic acid)

Phoebus Levene

incorrectly proposed the tetranucleotide hypothesis

(made other contributions to genetics, but those aren’t important)

tetranucleoide hypothesis

proposed by Phoebus Levene

DNA is composed of repetitions of the same sequence of four nucleotides (ATGC)

now known to be incorrect

four genetic properties of unknown genetic material

proposed by Frederick Griffith:

capable of replication

capable of storing information

complex enough to generate thousands of gene products

any changes in genetic material (mutations) should directly lead to changes in phenotype (now known that this is not always true)

Frederick Griffith

proposed the four properties of unknown genetic material

conducted the Griffith experiments, demonstrated genetic transformation in S. pneumoniae

the Griffith experiments

demonstrated genetic transformation in bacteria Streptococcus pneumoniae

strain IIIS (smooth) is virulent; causes fatal pneumonia in mice. Cells can be recovered from lungs of killed mice

strain IIR (rough) is nonvirulent; mice injected with it survive, not cells can be recovered from these mice.

Heat kills IIIS cells, but an unknown “transforming principle” survives and is capable of transforming IIR cells into IIIS cells

results:

mice injected with IIIS die, IIIS bacteria are recovered

mice injected with IIR survive, no bacteria are recovered

mice injected with heat-killed IIIS survive, no bacteria are recovered

mice injected with a mixture of IIIR and heat-killed IIS die, IIIS bacteria are recovered

transformation

a heritable change in a cell or organism brought about by exogenous DNA

the Avery, MacLeod, and McCarty experiments

identified Griffith’s transforming principle

IIIS cultures (in liquid media) were centrifuged, collected, heat-killed, lysed, and homogenized

enzymes and extractions eliminated with carbohydrates, lipids, and proteins

resulting solution had nitrogen:phosphorus ratio consistent with a nucleic acid, and was still capable of transforming IIR into IIIS cells

either proteins, RNA, or DNA were destroyed with enzymes

resulting mixtures were added to cultures of IIR cells

cultures with RNA and proteins destroyed contained transformed IIIS, culture without DNA did not

conclusion: IIIS DNA can transform IIR → IIIS cells

protease

enzyme that destroys proteins

used in the Avery, MacLeod, and McCarty experiments

ribonuclease (RNase)

enzyme that destroys RNA

used in the Avery, MacLeod, and McCarty experiments

deoxyribonuclease (DNase)

enzyme that destroys DNA

used in the Avery, MacLeod, and McCarty experiments

the Hershey & Chase experiments (1952)

showed that DNA is the infectious agent in bacteriophages

1) infect bacteria in radioactive media with T2 phages

2) infect cells in non-radioactive media with radioactive phages

3) after infection, separate phages from cells in a blender, centrifuge the suspension

³²P phages have radioactive DNA, ³⁵S phages have radioactive proteins

bacteria infected by ³²P phages incorporate radioisotope ³²P; radioactivity will be found in cells at the bottom of the centrifuge tube (the pellet)

bacteria infected by ³⁵S phages, the radioisotope stays outside of the cells; radioactivity will be found in the supernatant

conclusion: DNA, not protein, is the genetic material in phages

X-ray diffraction

used to elucidate the structures of molecules

steps:

crystals of substance are bombarded with X-rays through a lead screen. The X-rays get diffracted (bounce off of) the substance

the spacing of the atoms within the sample crystal determines the diffraction pattern. Diffraction pattern appears as spots on a photographic film

the diffraction pattern provides information about the structure of the molecule

Rosalind Franklin

credited with elucidating the helical structure of DNA

carried out X-ray crystallography of DNA fibers in 1952

photograph 51

diffraction profile was consistent with helical structure, possible double helix, with a sugar-phosphate backbone outside

worked in Maurice Wilkins’ lab at King’s College, London

Erwin Chargaff

studied the base-composition of DNA

formed Chargaff’s rules

the Watson-Crick model

DNA (deoxyribonucleic acid) is a double helix with antiparallel strands

strands are made up of a sugar + phosphate backbone and nitrogenous bases paired at the center by hydrogen bonds

alternating major and minor grooves

the length of one whole turn of the helix is ~10 base pairs (bp)

James Watson and Francis Crick “proposed” the double-helix model of DNA, published model in 1953, were awarded the Nobel Prize in physiology or medicine in 1962

ribose

a pentose sugar present in RNA, makes up the backbone along with phosphate

difference from deoxyribose: has an OH group at 2’C instead of a second H

deoxyribose

a pentose sugar present in DNA, makes up the backbone along with phosphate

difference from ribose: lacks an OH at 2’C, instead has a second H (only 2 OH groups directly connected to the pentose (exclude the OH connected to 5’C))

purines

one group of nitrogenous bases in nucleic acids

double carbon-nitrogen aromatic structures

in nucleic acids, include adenine and guanine

basic structure is depicted

pyrimidines

one group of nitrogenous bases in nucleic acids

single carbon-nitrogen aromatic ring structures

in nucleic acids, include cytosine, thymine, and uracil

basic structure is depicted

adenine (A)

a purine

found in both DNA and RNA

pairs with thymine in DNA

pairs with uracil in RNA

from basic purine structure, only difference is an added amine (NH2) at one C

guanine (G)

a purine

found in both DNA and RNA

pairs with cytosine in both DNA and RNA

from basic purine structure, a carbonyl (double bonded oxygen to carbon) is added to one C, the adjacent double bond is removed, an amine (NH2) is added to another C, and add Hs to Ns missing a double bond

cytosine (C)

a pyrimidine

present in both DNA and RNA

pairs with guanine in both DNA and RNA

from basic pyrimidine structure, add an amine (NH2) to one C, add a carbonyl (O double bonded to C) to another C, remove the adjacent double bond, and add Hs to Ns missing a double bond

thymine (T)

a pyrimidine

found only in DNA

pairs with adenine in DNA

from basic pyrimidine structure, add 2 carbonyls (O double bonded to C), remove the double bonds adjacent to both, add a methyl (CH3) to another C, and add Hs to Ns missing double bonds

uracil (U)

a pyrimidine

found only in RNA

pairs with adenine in RNA

from basic pyrimidine structure, add carbonyls (O double bonded to C) to 2 Cs, remove the adjacent double bonds, and add Hs to Ns missing a double bond

nucleoside

a unit made of a sugar and nitrogenous base

no phosphate

(think: no T in the name, no phosphaTe)

nomenclature of ribonucleosides

nucleotide (root) + either -sine or -dine (suffix)

suffix is -sine for purines, -dine for pyrimidines (both have a d in the name)

e.g. adenosine, cytidine, guanosine, uridine

nomenclature of deoxyribonucleosides

deoxy- (prefix) + nucleotide (root) + either -sine or -dine (suffix)

suffix is -sine for purines, -dine for pyrimidines (both have a d in the name)

e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxyuridine

nucleotide

a unit made of a sugar, nitrogenous base, and phosphate(s)

(think: has a T in the name, has phosphaTe)

consist of ribonucleotides and deoxyribonucleotides

nomenclature of nucleotides

same as nomenclature of nucleosides, then add mono/di/tri/etc-phosphate

e.g. uridine monophosphate (UMP), cytidine diphosphate (CDP), deoxythymidine diphosphate (dTDP), adenosine triphosphate (ATP)

phosphodiester bond

joins the 3’ C of one nucleotide and the 5’ C of the next through 2 ester linkages

has oxygens (OR groups) on both sides

link nucleotides in the polynucleotide chains of DNA and RNA

Watson-Crick base pairs

A-T and A-U: 2 hydrogen bonds form between the nucleotides

G-C: 3 hydrogen bonds form between the nucleotides

Chargaff’s rules

1) the proportion of A = proportion of T, and the proportion of G = proportion of C

2) there is an equal proportion of purines (A and G) and pyrimidines (C and T)

3) the proportion of C + G does not necessarily equal that of A + T

intramolecular hydrogen bonds

hydrogen bonds within a molecule

can form in most RNAs, e.g. transfer RNA (tRNA, because it is single-stranded)

results in endless possibilities for 3D structures

organization of DNA in eukaryotes

DNA is contained in nucleus as chromosomes

always present, but only visible (condensed chromosomes) during mitosis

organized in nucleosomes

adjacent nucleosomes pack together, form 30-nm chromatin fibers

higher order levels of organization results in the condensation of DNA into the visible chromosome beginning at prophase

nucleosome

the basic unit of organization of DNA in eukaryotic nuclei

each unit consists of 2 turns of DNA (147 bp) wrapped around 8 histone proteins and held together by a ninth histone (histone 1)

isolation and purification of DNA

procedure for isolation is similar to that used by Avery, MacLeod, and McCarty

purification: precipitation of DNA by the addition of ethanol, then the redissolving of the precipitate

done in preparation for enzymatic manipulation

nucleases

enzymes that break up polynucleotide chains

can be exonucleases or endonucleases

exonucleases

enzymes that remove nucleotides one by one from the ends of a polynucleotide chain

endonucleases

enzymes that cleave phosphodiester bonds within a polynucleotide chain

can produce sticky ends or blunt ends

restriction endonuclease

an enzyme that recognizes a specific base pair sequence in DNA and makes a cut at that site

the bp sequence is a restriction site with a palindrome sequence

restriction digestion of DNA

results in restriction fragments: small linear fragments with either complementary single-strand sticky ends (cohesive tails) or blunt ends

type of end depends on the restriction enzyme

e.g. HindIII makes sticky ends, PvuII makes blunt ends

e.g. can manipulate plasmids

cloning vector

e.g. pUC18

has a polylinker region that contains restriction sites for several restriction endonucleases

any foreign DNA introduced in this region of the plasmid will be replicated every time the cell divides

ways to introduce DNA into cells

electric shock

heat shock

microinjection

electrophoresis

a method for separating fragments of nucleic acids according to size

molecules migrate to agarose gel medium in electric field

because phosphate groups have negative charges, molecules migrate toward the positive end of the field

smaller molecules migrate faster (will end up closer to the positive end)

for nucleic acids, a dye is added to the gel. DNA fragments will appear as bands in the gel

preparation of an agarose gel

agarose gel: semisolid slab that has wells near one end for loading samples of nucleic acid

a tray with the gel is placed in an electrophoresis chamber, which is filled with buffer solution to cover the gel

DNA samples are loaded into the wells

electric field is applied; cathode (-) is placed at the sample end, anode (+) at the opposite end

ethidium bromide staining

used for electrophoresis of nucleic acids

fluorescent dye that becomes nested between the nitrogenous bases

can be excited by UV light to show bands of migrated DNA

presentation of DNA sequences

the sequence of nucleotides of only one strand is shown

e.g. present only the 5’ to 3’ strand, not the 3’ to 5’ strand as well

(the other strand is complementary, so it would just be the presented strand in backwards order)

conclusion of the Avery, MacLeod, and McCarty experiments

because only DNase destroyed the transforming substance, the transforming substance if DNA

the infectious agent in phages is

DNA

High G-C content DNA have higher

density and melting point

nucleoside

sugar + nitrogenous base

nucleotide

sugar + nitrogenous base + phosphate(s)

deoxyribose lacks the

2’ OH group

how do you number the carbons in the pentose sugars

1’ to 5’, right to left

what is the nucleosome core particle

eight histone proteins