GENETIC MATERIAL

CHAPTER 8

Central Dogma of Molecular Genetics

Early Studies

• Beginning with the earliest observations concerning heredity, genetic material was assumed to exist

• Until the 1940s proteins were considered by geneticists to be the best candidates– Very abundant in cells and did nifty things– Nucleic acids were similar, boring and just a

couple of nucleotides connected to each other…

Discovery of DNA

• 1868 by Friedrick Miescher, a Swiss chemist– Called in nuclein since it was from the nucleus– Had large amounts of phosphorous and no

sulfur so was very different than protein

First Structure

• By 1910 actual components known (nucleotides)– Phoebus Levene

proposed a tetranucleotide structure for DNA

• Tetranucleotide repeat of ATCG• Own data showed nucleotides not in 1:1:1:1 ratio

Differences “probably experimental error…”

So…

• If DNA was a single covalently bonded tetranucleotide structure then it couldn’t easily encode information

• Proteins, on the other hand, had 20 different amino acids and could have lots of variation

• Most geneticists focused on “transmission genetics” and passively accepted proteins as being the likely genetic material

First Real Break

• 1927, Frederick Griffith

• Studied Pneumococcus (then became Diplococcus pneumoniae, then became Streptococcus pneumoniae)– IIR strain was avirulent and lacked a

lipopolysaccharide (LPS) capsule, growing in rough-shaped colonies on a plate

– IIIS strain was virulent, possessed a lipopolysaccharide capsule and could kill mice, and made round colonies

Frederick Griffith

• The Experiment – Inject mouse with strain S mouse dies– Inject mouse with strain R mouse lives– Inject with heat-killed strain S mouse lives– Inject with h-k S and live R mouse dies, and live S

strain can be recovered from dead mouse

• Griffith concluded that the live R had been transformed to S by picking up the genetic material encoding the LPS from the dead S and using that material to repair the damaged/lost gene in the R strain

Griffith’s Experiment

• Called the material in the dead S cells that allowed for the RS transformation the transforming principle

• First assay for the genetic material

Avery, McCarty and MacLeod

• After 10 yrs of effort published work using Griffith’s approach to assay for the genetic material– Used

• Cell-free extract of S cells• From 75 liters of cell culture obtained 10-25 mg of “active

factor• Proteases, RNases, DNases, etc.

• “The evidence presented supports the belief that a nucleic acid of the desoxyribose type is the fundamental unit of the transforming principle of Pneumococcus Type III”

Avery, McCarty

and MacLeod

Harriet Taylor

• 1949 follow-up• Studied strain R and strain ER (extremely

rough)• Showed DNA from R could convert ER strains

to R strains– and then DNA from S strains could convert R to S

strains

• Conclusion: R strains could be both donor and recipients in transformation experiments

Hershey Chase Experiment

• Alfred Hershey and Martha Chase, 1952

• Evidence that DNA is the genetic material

• Simple model system using T2 bacteriophage and radioactive materials

Life Cycle of T-Even Phage

• Phage made of DNA and protein– What enters cell

and allows production of new phage?

Hershey Chase Experiment

• T2 Phage, E. coli, and 35S, Waring blender– 32P04 goes into DNA– 35S04 goes into proteins

• Experiment– Grow phage on cells cultured in 32P04 and 35S04

– Infect new cells (not radioactive) with radioactive phage– After various times place in Waring blender, centrifuge

and measure radioactivity in cells plus plate them out to determine whether successfully infected by phage

– Allow some to complete life cycle and measure radioactivity levels of progeny phage

Hershey-Chase Experiment

• Time course also reveals that entry of 32P into cells correlates with successful infection

Indirect Evidence for Eukaryotes

• DNA found “only” in nucleus, proteins all over cell

• DNA in chromosomes

• Ploidy correlated with DNA content

More Indirect Evidence: Mutagenesis

• Action spectrum of UV light for mutagenesis correlates well with the absorption spectrum of DNA– UV light of 260 nm most mutagenic– DNA absorption maximum is 260 nm– Protein absorption maximum is 280 nm

Action and Absorption

Spectra

RNA as Genetic Material

• Fraenkel-Conrat and Singer, 1956

• Tobacco Mosaic Virus (TMV) and Holmes Ribgrass Virus (HRV)– Closely related plant viruses made of an RNA

molecule encased in a spiral of protein– One coat protein could encapsulate the other

RNA and still function properly during infection

RNA as Genetic Material

RNA Can Replicate

• Pace and Spiegelman, 1965, 1966

• Phage Q• Isolated an RNA replicase enzyme that

could replicate the Q chromosome in vitro– No DNA involved

Reverse Transcription

• Retroviruses (e.g. HIV, RSV)– RNA chromosomes– Convert to DNA by reverse transcriptase– Insert DNA into host chromosome– Transcribe new RNA copies

Nucleic Acid Structure• DNA is a nucleic acid composed of nucleotides

– Nucleotides have a nitrogenous base, a pentose sugar and a phosphate group

– Bases are either pyrimidines (cytosine and thymine in DNA or C and uracil in RNA) or purines (adenine and guanine)

– Pentose sugar is either deoxyribose (DNA) or ribose (RNA)

– A base plus a sugar is a nucleoside, add phosphate for a nucleotide (nucleotides named by nucleoside plus number of phosphates – adenosine diphosphate)

– Sugar on C-1’ position, phosphate commonly on C-5’

Components of Nucleic Acids

• Purines• Pyrimidines• 5-carbon

sugar• phosphate

Nucleosides and Nucleotides

Nucleoside Diphosphates and Triphosphates

Polynucleotides

• Nucleotides of a single strand connected by covalent 5’-3’ phosphodiester bond

• Following Levene’s tetranucleotide hypothesis it was clearly shown that bases were not present in equimolar quantities and that DNA molecules were in fact quite large

Phosphodiester Bonds

• Phosphate is from phosphoric acid

• Hydroxyl groups on sugars represent alcohol

• Acid plus alcohol given ester– Phosphate reacts with

two –OH groups

Structure of DNA

• Structure of DNA should reveal how it works as the genetic material

• Intense study from 1940-1953

• Chargaff, Wilkins, Franklin, Pauling, Watson, Crick and more…

• First to elucidate the correct structure gets the big one

Erwin Chargaff

• 1949-1953

• Digested many DNAs and subjected products to chromatographic separation

• Results– A = T, C = G– A + G = C + T (purine = pyrimidine)– A + T does not equal C + G

• Members of a species similar but different species vary in AT/CG ratio

Franklin and Wilkins

• X-ray diffraction analysis of DNA crystals– Originally by William Astbury (1938)who

detected a periodicity of 3.4 angstroms (1947)• Pauling used data to propose a triple helix

– 1950-1953 Franklin (in Wilkins’ lab) confirmed 3.4 periodicity and noted uniform diameter of 20 angstroms (2 nm)

• Proposed no definitive model

X-ray Crystallography

of DNA

• Franklin and Wilkins

Watson and Crick

• 1953 propose double helix model– Right-handed double helix– Chains antiparallel– Bases lie flat, perpendicular to long axis of chain– Bases pair by hydrogen bonds, A with T and C with G

• Two strands are complementary

– 10 bases per turn (34 angstroms)• Now known to be 10.4 or 34.6 degrees turn per bp)

– Has a major and minor groove– Is 20 angstroms in diameter

DNA Double Helix

Right vs. Left Handed Helices

Base Pairing

• Hydrogen bonds– reversible

– Individually weak electrostatic bonds but collectively can be strong

Impact

• Article in Nature– “It has not escaped our notice that the specific

pairing we have postulated immediately suggests a possible copy mechanism for the genetic material”

• Second paper 2 months later describes semiconservative replication and that mutations must change bases in DNA (information encoded in the bases and their order)

• DNA became the genetic material…

Alternative Forms of DNA

• DNA can exist in several conformational isomers– B form is the “normal” conformation– A form is found in high salt

• Probably not biologically relevant

– D and E forms (8 and 7 bp/turn respectively)• DNA segments lacking guanine

– Z form• Left-handed helix and 12 bp/turn (Z for zigzag)• C-G base pairs only

– P form• Phosphates to inside and bases more to outside

• Are P and/or Z biologically relevant???

Conformational Forms of DNA

Structure of RNA• Ribose for deoxyribose, uracil for thymine

• RNA tends to be single stranded– Can fold back to have secondary structure– Can be double stranded in some phage/viruses

• Major classes of RNA– Ribosomal RNA– tRNA– mRNA– But there are several others…

Major Classes of RNA…but there are more

• S is for the Svedberg sedimentation coefficient

Other RNAs

• To be discussed in later chapters– snRNAs– Telomerase RNA– siRNAs– Antisense RNAs

Nucleic Acid Characterization

• Absorption Spectra– Absorb light in ultraviolet range, most strongly

in the 254-260 nm range• Due to the purine and pyrimidine bases

• Useful for localization, characterization and quantification of samples

Nucleic Acid Characterization

• Sedimentation and density– Can be characterized by sedimentation velocity

(Svedberg coefficient, S)• Sedimentation velocity centrifugation

• Related to MW and shape

– Or by buoyant density• CsCl (DNA) or CsSO4 for RNA

• Sedimentation equilibrium centrifugation

Buoyant Density

Centrifugation

Base Composition vs. Density

• G-C base pairs are more dense than A-T pairs

Denaturation of Nucleic Acids

• Denaturation involves the breaking of hydrogen bonds– Disrupts the base stacking in the helix and lead to

increased absorbance at 260 nm• Hyperchomic shift

• By increasing temperature slowly and measuring absorbance at 260 nm as melting profile can be generated– Temperature for midpoint of denaturation is called

the Tm

Thermal Denaturation• Increased

G+C gives increased Tm – 3 vs. 2

hydrogen bonds

• Increased ionic strength also increases Tm

Hybridization

• After nucleic acids are denatured they can be allowed to reform base pairs with complementary molecules– Molecular hybridization– Close but not perfect match required

• stringency

– Can involve DNA:DNA or DNA:RNA– FISH, Southern transfer (blotting) and DNA

microarray analyses involve hybridization

Hybridization

Fluorescent in situ Hybridization

• FISH• Use DNA or RNA probes

for hybridization– Originally radioactive

– Now biotin and fluorescent dyes

• Cells/chromosomes fixed to slide before hybridization

• Can detect single copy genes

Reassociation Kinetics

• Denatured DNA duplexes can reassociate with complementary strands to reform duplex – Chemical reaction, rate depends upon

conditions• including substrate concentration

Reassociation Kinetics

Reassociation Kinetics

• DNA concentration is routinely measured in micrograms per ml (mass/volume)– But here the relevant concentration is copies of

complementary DNA (not mass) per unit volume

– And this depends upon both the mass per volume and the size of the genome being studied

Reassociation Curves of Different DNAs

Genome Size vs. C0t1/2

C0t Analyses

• Previous curves were for genomes generally lacking repetitive sequence regions– Al or nearly all sequences present at one copy

per genome

• What happens to the C0t analyses when genomes have repetitive sequences?– Single copy, middle and highly repetitive

C0t Analyses

Gel Electrophoresis

• Agarose or polyacrylamide gels

• DNA is negatively charged and migrates toward positive pole when placed in an electric field

• Smaller fragments move through the gel matrix more quickly and therefore migrate faster per unit of time

• Extremely common method for characterizing and purifying DNA fragments– Including DNA sequencing procedures

Gel Electrophoresis