L8 Biol261F2014DNA Strfunct.ppt

8/9/2019 L8 Biol261F2014DNA Strfunct.ppt

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DNASTRUCTUREis key to understanding:

(1) replication;

(2) inheritance;

(3) allele diversity;(4)mutation; and

(5) protein

expression and variation

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1850-1950: In historical order, major landmarks in genetics

(1)Mendel: genes hereditary particles,gene allelescorrelated with character states,

tested in breeding designs and the logic extended to pedigrees,other allelic and genetic interactions, complementation, andmutant screens.

(2)Genes are on certain chromosome locations (T.H. Morganand A. Sturtevant 1910-1920), and crossover involves breakageand rejoining (H. Creighton and B. Mclintock 1931).

(3) Strain types (genes) can be transformed into different types bythe presence of other dead strains (Griffiths 1928).

(4) One gene related to one enzyme in pathway analyses - G. W.Beadle and E. L. Tatham (1941) - then one polypeptide.


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DNA : discovery of the

genetic material.

In 1928 Frederick Griffiths demonstrated the existenceof a

transforming principle

that changed the formand function of a bacterial strain type ofStreptococcus

pneumoniae into another type.

A non-virulent strain that formed rough-looking colonies (R)was transformed, to a virulent (geno)type associated withsmooth (S) colonies. However, this could only be attributed to a

principle

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Transformation principle: unexpected results and the

empiricalvalue of experimental controlsin an in vivo study.Heat 60 oC =transformation; no transformation > 80 oC ,freezing & thawing or old cultures

In 1931, R. Sia and M. Dawson performed the same

experiment in vitro, in liquid culture, showing .

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In 1944 the laboratory of OswaldAvery, ColinMacLeodandMaclynMcCarthyidentified the biochemical classof the

transforming principle, following Sia and Dawsonsexperiment.

First they developed an assay measuring the conditionswherein R cells of an optimal strain could be reliablytransformed by a volume of a cell free extract of S cells.

Using selective elimination of the major biochemicalcategories, They showed that only DNA deoxyribonucleicacid, was capable of replicating the transforming principaleffect.

Surprisingly, it was not in a protein, not a carbohydrate, not a

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Beginnings of molecular genetics


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In 1952 Alfred Hershey and Martha Chase confirmed thatDNA was the genetic material, at least in virus.

At that time the method of phage replication was not known,but it was known that the T2 phage was approximately 50%protein, 50% DNA.

They used radioactive isotopes of phosphorus and sulphur

to label components of T2 phage.

32P labelled the DNA, 35S labelled the protein.

Only the 32P , i.e. DNA, of T2 appeared toenter the bacterialcell mediating the infection and replication of the phage.Importantly, after the cells lysed and the progeny phage emergedmany were labelled with 32P.

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The % A and C or G and T in the DNA from different species wasdifferent.

But the % A always is (approximately) equal to the % T and the % G isalways (approximately) equal to the % C.

Chargaffs rule: A ~ T and G ~ C, but,

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J. Watson on R. Franklin The real problem was Rosy.There was no denying she had a good brain. If she could

only keep her emotions under controlThe thoughtcould not be avoided that the best home for a feministwas in another persons lab.pp 15 The Double Helix

Chargaff I told them all I knew. If they had heardbefore about the pairing rules, they concealed it. But asthey did not seem to know much about anything, I was notunduly surprised. I mentioned our early attempts to

explain the complementarityrelationships by theassumption that, in the nucleic acid chain, adenylicwas always next to thymidylic acid and cytidylic nextto guanylic acid...I believe that the double-stranded

model of DNA came about as a consequence of our


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What are the requirements for genetic material explaining the inheritance of molecules,physiology, morphology, behaviour etc.

A codethat is: (1)stable (2) replicable (3) expressible (4)can evolve

In April 1953, Watson and Crick proposed a structural model inNature:

We propose

the double helix, (stable structure)

complementary base pairing (expressible) and

a mechanism for DNA replicationby unwinding and separating

Followed by a scale model(1954 Proc. R. Soc. (A): 80 - 96) they also proposed a mechanis

for mutation (can evolve)

Several years later, Crick et al. (1961) provided evidence that the code involved triplet base

sequences.

The Problem Watson and Crick took on 15


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DNA Stability

(1)A large number of hydrogen bonds joiningstrands.

(2)Each strand has a chain of covalently bonded,

phosphate-nucleosideunits (backbone).

(3)A-T, C-G pairs (purine -pyrimidine) samediameter along the length, no bulgesin the helix.

(4)Charged groups face outward into water-

interact strongly.

(5)

Helical structure means the inner H -bondednucleotides are protected from interacting withwater

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2-dimensional view of DNA

Note:

!C=G and A=T nucleotide pairingdiffers in the number of H bonds

!

Deoxyribose-phosphodiesterbackbone.

!directionality of each strand (5 to 3).The two strands are antiparallel,

one going 3 to 5, the other 5 to 3

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Hydrogen bonding between nucleotide pairs.Individual hydrogen bonds are much weaker than covalent

bonds, but 3 bonds >2, and there are many of them.

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3-5 covalent

phosphodiester bondbetween twonucleosides(deoxyribose + base)Forms the backbone

of each strand

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The major (22A across) and minor grooves (12 A) areimportant for proteins that regulate gene expression.

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There was one other importantlevel of organization in functionalDNA, they are supercoiled no free

ends. Linear chromosomes areanchored to a protein scaffold.

Almost all organisms havenegatively supercoiled DNA, and

many biological functions can onlybe carried out with negativelysupercoiled DNA (replication ,recombination gene expression andregulation) in both bacteria andeukaryotes.

Supercoiling produces tension inDNA structure, which is probablyimportant in replication

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Twist -#helical turns.Writhe -# times double helix crosses

itself


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The structure of DNA presents a mechanism to account fortwo key functionsof genetic material.

1. Complex information storage.

The vast variety of A, T, C and G sequence along aDNA strandcan contain complex information.

2. Replication.

The double stranded complementary base structure

facilitates rapid and accurate SEMI CONSERVATIVEduplicationof the DNA.

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1958: Mathew Meselson and Franklin Stahl testedthree possible models of replication.

(1) They grew E. coliin the presence of a heavy isotope ofnitrogen, 15N. This produced DNA of high density.

(2) They followed this with growth in normal 14N for one cycleand(3) A second cycle of replication in 14N.

DNA density was measured by centrifuging in an ultracentrifugein a cesium chloride gradient.

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What are the requirements for genetic material explaining the inheritance of molecules,

physiology, morphology, behaviour etc.

A codethat is: (1)stable (2) replicable (3) expressible (4)can evolve

In April 1953, Watson and Crick proposed a structural model in Nature:

We propose

the double helix, (stable structure)

complementary base pairing (expressible) and

a mechanism for DNA replicationby unwinding and separating

Followed by a scale model(1954 Proc. R. Soc. (A): 80 - 96) they also proposed a mechanism

for mutation (

can evolve

)

Several years later, Crick et al. (1961) provided evidence that the code involved triplet base

sequences.

The take-home message here is that the structure of DNA, isstable, replicable, expressible, mutable and determines theprotein code , or

structure and function are inseparable

L8 Biol261F2014DNA Strfunct.ppt

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