Lect 2- DNA and Molecular Genetic

8/11/2019 Lect 2- DNA and Molecular Genetic

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Molecular GeneticsChromosome

DNA

Nucleotides

Nucleus

Cell

2

DNA DNA is often called

the blueprint of life. In simple terms,

DNA contains theinstructions formaking proteinswithin the cell.


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Why do we study DNA ?

We study DNA formany reasons,e.g.,

its centralimportance to alllife on Earth,

medical benefits

such as cures fordiseases,

better food crops .

Cellular composition

DNA is contained innucleus of cell

Phospho-lipids andproteins combined toform cell membrane

Lipids are fats

Our genes a re on ourc h r o m o s o m e s .

C h r o m o s o m e s a re m a d e u pof a chem ical cal led DNA.


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Genetic material of cells

GENES units of genetic material thatCODES FOR A SPECIFIC TRAIT

Called NUCLEIC ACIDS

DNA is made up of repeating moleculescalled NUCLEOTIDES

A HISTORY OF DNA

Discovery of the DNA double helix

A. Frederick Griffith Discovers that a factorin diseased bacteria can transform harmlessbacteria into deadly bacteria (1928)

B. Rosalind Franklin - X-ray photo of DNA.(1952)

C. Watson and Crick - described theDNA molecule from Franklins X -ray.(1953)


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Watson and Crick (1953) Used data of M.H.F. Wilkin s

and Rosal ind Frankl in ,early 50s

Wilkins and Franklin studiedthe structure of DNA crystalsusing X-rays.

The X pattern suggested thestructure of DNA was a helix.

Distance between the two backbones of DNAis constant along the length of the molecule

Used data of Erwin Chargaff ,

1940s and early 50's Chargaffs Rule : His datashowed that in each species,the percent of A equals thepercent of T, and the percent ofG equals the percent of C.

8

The Shape of the Molecule

DNA is a very longpolymer.

The basic shape is

like a twisted ladderor zipper. This is called a

do ub le he l ix .


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DNA Nucleotide

OO=P-O

O

PhosphateGroup

N

Nitrogenous base(A, G, C, or T)

CH2

O

C1C4

C3 C2

5

Sugar(deoxyribos e)

12

Four nitrogenous bases

Cytosine C

Thymine T Adenine A Guanine G

DNA has four different bases:


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Two Kinds of Bases in DNA

Pyr imidines aresingle ring bases .

Purines aredouble ringbases.

C C

C C

N

N

O

N

C C

C C

N

N

N

NNC

14

Thymine and Cytosine arepyrimidines

Thymine and cytosine each have onering of carbon and nitrogen atoms.

C

C

C

C

N

N

O

N

cytosine

C

C

C

C

N

N

O

O

thymine

C


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Adenine and Guanine arepurines

Adenine and guanine each have tworings of carbon and nitrogen atoms.

C

C C

C

N

N

N

Adenine N

NC

C

C C

C N

NO

N

Guanine N

NC

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Two Stranded DNA Remember, DNA

has two strandsthat fit togethersomething like azipper.

The teeth are thenitrogenousbases but whydo they sticktogether?


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C C

C

C

N

NO

N

C C

C

C

N

N

O

N

N N C

Hydrogen Bonds

The bases attract eachother because ofhydrogen bonds.

Hydrogen bonds are weakbut there are millions andmillions of them in asingle molecule of DNA.

The bonds betweencytosine and guanine areshown here with dottedlines

18

Hydrogen Bonds, cont. When making

hydrogen bonds,cytosine alwayspairs up withguanine

Adenine alwayspairs up withthymine

Adenine is bondedto thymine here

C

C

C

C

N

N

O

O

C


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Chargraffs Rule:

Adenine and Thyminealways join together

A T

Cytosine and Guaninealways join together

C G

Genetic Diversity Different

arrangements ofNUCLEOTIDES in anucleic acid (DNA)provides the key toDIVERSITY amongliving organisms.


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The Code of Life

The code of the chromosome is theSPECIFIC ORDER that bases occur.

A T C G T A T G C G G

DNA is wrapped tightly aroundhistones and coiled tightly to form

chromosomes


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Primary, Secondary, and Tertiary Structure of NucleicAcids

The primary structure is the sequence of nucleosidemonophosphates (usually written as the sequence ofbases they contain).

2. The secondary structure refers to the shape a nucleicacid assumes as a result of the primary structure . B-DNA, A-DNA, and Z-DNA are forms of secondarystructure . B-DNA is the form that predominates in theaqueous environment of the cell.

3. Tertiary structure refers to large-scale folding in a linearpolymer that is at a higher order than secondarystructure . The tertiary structure is the specific three-dimensional shape into which an entire chain is folded.

Phosphodiester Bonds

Adjacent monomer units in nucleic acids are connected viaphosphate groups attached to the hydroxyl on the 5' carbonof one unit and the 3' hydroxyl of the next one. This linkageis called a phosphodiester bond.

1. Phosphodiester bonds in nucleic acids are very stable tohydrolysis in the absence of a catalyst (such as an acid or anuclease).

2. Synthesis of a phosphodiester bond in nucleic acidsrequires energy input. As a result, the nucleosidemonophosphates in nucleic acids are built up fromhydrolysis of nucleoside triphosphates . Cleaving apyrophosphate from a nucleoside triphosphate yields anucleoside monophosphate and enough free energy tomake the formation of polynucleoside monophosphates (i.e.,polynucleotides) thermodynamically favorable.
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DNA Replication

DNA must be copied

The DNA molecule produces2 IDENTICAL newcomplementary strandsfollowing the rules of basepairing ( Chargraffs Rule) :

A-T, G-C

Each strand of theoriginal DNA serves asa template for the newstrand

Threemodels ofDNAreplication

1. Conservative From one parentaldouble-stranded DNA, two"daughter" double-stranded DNAsare made. One contains two newstrands and the other contains bothof its original strands.

2. Semi-conservative From oneparental double-stranded DNA, twodaughter double-stranded DNAs aremade. Each daughter DNA containone parental DNA strand and onenewly made strand.

3. Dispersive From one parentaldouble-stranded DNA, two daughterdouble-stranded DNAs are made.Each strand in the daughtermolecules contains portions of oldand newly synthesized material.


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DNA Replication Overview DNA replication is an essential aspect of cellular and viral reproduction.

Replication of a double-stranded DNA results in two double-strandedDNAs as products. Some important general points about DNAreplication are as follows:

The mechanism ofreplication is semi-conservative--each newlymade strand is copied fromone of the parental strandsand the products of replicationare two molecules, eachcontaining one parental strand

and one newly synthesizedstrand.

DNA replication intermediates contain "forked"structures at the site ofreplication ( Figure ).

Replication is orderly and sequential--it begins at a fixed point (calledan origin) and closely follows parental duplex unwinding.

DNA replication uses deoxyribonucleoside-5'-triphosphates (dNTPs) tobuild the DNA chains.

DNA replication is discontinuous--synthesis of one strand (called thelagging strand) lags behind the other (called the leading strand) andoccurs in pieces called Okazaki fragments ( Figure ). Replication of theleading strand is continuous ( Figure ).

Replication is exceedingly accurate--far more accurate than any otherenzyme-catalyzed process.

Replication can be broken down into three processes--initiation,

elongation, and termination. Multiple proteins are required for DNA replication at a replication fork.These include DNA polymerases, single-strand DNA binding proteins,helicases, primase, topoisomerases, and DNA ligase. Some of theseare multisubunit protein complexes.

DNA polymerase catalyzes the chemical reaction of DNA synthesis. DNA chain growth ( replication ) proceeds only in the 5' to 3' direction.
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The Okazaki model.

Details of lagging strandsynthesis

Initiation of DNA Replication

DNA replication is initiated specifically from an origin. Initiation appears to be the major target for the control of replication. Tworequirements for replication initiation are as follows:

1. A nucleotide sequence that specifically binds initiation proteins, and2. A mechanism that generates a primer terminus for DNA polymerase

to extend.

The two most straightforward ways to generate a primer terminus at theorigin are as follows:

1. Nicking a strand of the parental duplex to expose a 3' hydroxylterminus

2. Synthesizing an RNA primer to expose a 3' hydroxyl ribonucleotideterminus.


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Figure shows a generalized replication scheme for single strand phageDNA. The main points are as follows:

1. The single plus (+) strand genome enters cells. It serves as thetemplate for the synthesis of the complementary minus (-) strand.

2. The duplex, called RFI (Replicative Form I), has superhelical turnsintroduced into it.

3. A site-specific initiation protein nicks the DNA at a specific sequence inthe (+) strand and attaches to its 5' end.

4. Replication proceeds via extension of the 3' end. The 5' end isdisplaced in a rolling circle mechanism and single strand DNA bindingproteins attach to the displaced strand.

5. 5' to 3 extension continues.

6. Completion of one full circle of replication causes a protein to nick andrelease the original plus strand and generate a new duplex (calledRFII) containing the original minus strand.

Figure : Replication scheme for single-strand phage DNAs.
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Eukaryotic DNA Replication

Mechanisms - The basic mechanisms of DNA replication are quite similar in eukaryotes and prokaryotes.

DNA replication is semiconservative and is continuous onone strand and discontinuous on the other.

As in prokaryotes, eukaryotic replication entails theassembly of short RNA primer molecules, elongation fromthe primers by a DNA polymerase , and (on thediscontinuous strand) ligation of Okazaki fragments.

A significant difference in eukaryotic and prokaryotic DNA

replication is in the smaller size of the Okazaki fragmentsin eukaryotic cells - about 135 bases long, or about thesize of the DNA on a nucleosome .

Replication ForkReplication of DNA occurs at a molecular junction that is usually drawnschematically as a fork and is hence called a replication fork .Figure depicts a replication fork in E. coli along with many of theproteins that participate in DNA replication. The figure shows thatleading strand and lagging strand replication occur on opposite strandsat the same replication fork and that replication proceeds for bothstrands in the 5' to 3' direction.
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The terms in Figure are described below:

1. Topoisomerase - an enzyme that relieves the torsionalstress that arises ahead of the replication fork when the

helicase enzyme unwinds the DNA strands (Figure A ,Figure B ).

Fig. A: Action of a type I topoisomerase.

Fig. B: Action of a type II topoisomerase.

2. DNA polymerase - catalyzes the chemical reactions for

polymerization of nucleotides.Eukaryotic DNA PolymerasesMammalian cells contain four distinct DNA polymerases , while yeast cells contain at least five - , , , ,and . A short summary of the properties of each enzyme is as follows: - Distinctive for containing a primase activity, it is also highly sensitive to an inhibitor called aphidicolin .

Functions in lagging strand synthesis. - It has low processivity (i.e., it does not polymerize DNA for long periods of time). Functions in DNA

repair. Low sensitivity to aphidicolin. - A mitochondrial DNA polymerase. Low sensitivity to aphidicolin. - It may be the principal leading strand polymerase. Requires a protein called proliferating cell nuclear

antigen (PCNA) to carry out highly processive DNA synthesis in vitro . PCNA functions like the clamp ofE. coli DNA Polymerase III holoenzyme .

- Its function is not yet completely clear.
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3. Helicase - ( Figure ) an enzyme that unwinds DNA strands ahead ofthe DNA polymerase. Each strand of parental DNA has it ownhelicase. The one associated with the lagging strand is complexedwith primase as part of a unit called the primosome.

4. Primase - an enzyme that copies a DNA template strand bymaking an RNA strand complementary to it. The RNA serves as apriming site where DNA polymerase can begin to synthesize aDNA strand.

Figure: A model for helicase action.

5. Primosome - a complex containing aprimase and helicase. It helps to initiateDNA replication by synthesizing anRNA primer and to elongate it byunwinding the strands in advance ofthe replication complex.

6. Single-strand DNA-binding protein(SSB) - binds single-stranded DNA tostabilize it so that the hydrogen-bonding surfaces of the DNA bases arespatially oriented toward the incomingnucleotides ( Figure ).

Figure : gp32 facilitation ofboth denaturation andrenaturation of DNA.
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7. Sliding clamp - a protein dimer that encircles the DNA strand and helps holdthe DNA polymerase to the DNA strand.

8. RNA primer - a preexisting nucleic acid strand of RNA on which DNAreplication is continued. The initiation of DNA synthesis requires a preexistingnucleic acid strand, so RNA primers are frequently used for this purpose. RNAprimers are made by the primase enzyme.

9. Okazaki fragment - short discontinuous stretches of DNA arising fromreplication on the lagging strand. Okazaki fragments are named for thebiochemists who discovered them.

10.DNA polymerase I and DNA ligase - the two enzymes that assemble shortOkazaki fragments into a single continuous strand. DNA polymerase I has acatalytic activity that can remove RNA primers then replace them with DNA.DNA ligase catalyzes the covalent joining of the individual pieces of the laggingstrand.

11.Leading strand - the strand of DNA at a replication fork that replicatescontinuously.

12.Lagging Strand - the strand of DNA at a replication fork that replicates inpieces (Okazaki fragments).

1. Why is replication necessary?

2. When does replication occur?

3. Describe how replication works.

4. Use the complementary rule tocreate the complementary

strand:

A---?G--- ?C--- ?T--- ?A---?G--- ?A---?G--- ?C--- ?A---?G--- ?T--- ?

Replication Quiz


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1. Why is replication necessary?

So both new ce l l s wi l l have the cor rec tDNA2. When does replication occur?During interph ase (S phase) .3. Describe how replication works.Enzym es unz ip DNA and com plementary

nucleo t ides jo in each or ig ina l s t rand .4. Use the complementary rule to

create the complementary strand:

A---TG--- C

C--- GT--- AA---T G--- CA---T G--- CC--- GA---T

G--- CT--- A

Replication Quiz

(1961) Watson & Crick proposed DNA controlled cell function by

serving as a template for PROTEIN structure.

3 Nucleotides = a triplet or CODON(which code for a specific AMINO ACID)

AMINO ACIDS are the building blocksof proteins.

Refreshment


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Genetic Concepts

Gene sequence of DNA which istranscribed into RNA rRNA, tRNA or mRNA

Locus the position on a chromosome ofa particular DNA sequence (gene)

G Locus gene for color

W Locus gene for shape

DNA Transcription

DNA can unzipitself and RNA nucleotides matchup to the DNAstrand.

Both DNA & RNAare formed fromNUCLEOTIDES andare called NUCLEIC acids.


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Transcription

DNA is copied to RNA T is changed to a U So then A bonds with a U

(Uracil) Proceeds in the 5 -3

position mRNA leaves nucleus

as a copy and codes for

an amino acid(translation)

ProteinBiosynthesis


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DNA Translation The cell uses information from

messenger RNA to produceproteins

Function of mRNA, tRNA,Ribosomal ( Protein-Synthesizing Machines) ?

A ribosome is composed of several different ribosomal RNA (rRNA) molecules and more than 50 proteins, organized into alarge subunit and a small subunit. The proteins in the twosubunits differ, as do the molecules of rRNA. The small

ribosomal subunit contains a single rRNA molecule, referred toas small rRNA; the large subunit contains a molecule of largerRNA and one molecule each of two much smaller rRNAs ineukaryotes . The ribosomal subunits and the rRNA molecules arecommonly designated in svedbergs (S), a measure of thesedimentation rate of suspended particles centrifuged understandard conditions. complex structures, which physically movealong an mRNA molecule, catalyze the assembly of amino acidsinto protein chains. They also bind tRNAs and various accessorymolecules necessary for protein synthesis.

Translation

occurs within thecytoplasm of cell

tRNA transfer RNA decodes information

from mRNA toproduce amino acids

3 codons translate toan amino acid
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Codons

The term codon refers to a sequence of three nucleotides in amessenger RNA ( mRNA ) that specifies the incorporation of a specific

amino acid into a protein. The relationship between codons and theamino acids they code for is called the genetic code . The process ofconverting mRNA sequence information to the amino acid sequence of aprotein is called translation . An anticodon is a complementary 3 basesequence in transfer RNA ( tRNA ).Not all codons are used with equal frequency. In fact, there is aconsiderable amount of variation in the patterns of codon usage betweendifferent organisms.

Anticodon

An anticodon is a sequence of three nucleotides in a transfer RNA(tRNA ) that is complementary to a codon of messenger RNA ( mRNA ).The relationship between codons and the amino acids they code for iscalled the genetic code . The process of converting mRNA sequenceinformation to the amino acid sequence of a protein is called translation .

Amino Acid Activation

Requires amino acids tRNAs aminoacyl-tRNA synthetases

ATP, Mg 2+ Formation on an aminoacyl-tRNA

amino acid + aminoacyl-AMP + PP i

aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

amino acid + ATP + tRNA

aminoacyl-tRNA + AMP + PP i

ATP
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Amino Acid ActivationPP i

A-r ib- O- P- OPOPO -

O -

O

O -

ATP

+ - O-C- CH- RO

N H 3+

Amino acid

A-rib-O-P- O- C-CH- R

O

N H3

+

An aminoacyl-AMP

Step 1:O

O -

O

O -

OAMP

H

A

HOH HO

H HO

tRNA

+

Transfer RNA

H

A

HO HO

H HO

tRNA

C-CH- R

N H 3+

O

An aminoacyl-tRNA

Step 2:

A-rib-O-P- O- C-CH- RO

N H 3+

An aminoacyl-AMP

O -

O


This two-stage reaction allows selectivityat two levels the amino acid: the aminoacyl-AMP remains

bound to the enzyme and binding of thecorrect amino acid is verified by an editingsite in the tRNA synthetase

tRNA: there are specific binding sites ontRNAs that are recognized by aminoacyl-tRNA synthetases. Figure 11.7 (next screen)shows the locations of the recognition sites forthe tRNAs for various amino acids


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Ribbon diagram of tRNA tertiary structure

Chain Initiation

Requires fmet-tRNA fmet initiation codon (AUG) of mRNA 30S ribosomal subunit

50S ribosomal subunit initiation factors IF-1, IF-2, and IF-3 GTP, Mg 2+


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Chain Initiation In prokaryotes, the initial N-terminal amino

acid is N-formylmethionine (fmet)

Met + tRNA fmetMet-tRNAsynthetase

(ATP)Met-tRNA fmet

Formyl - FH 4

FH 4

Met-tRNA fmet

formyltransferase

CH 3 -S-CH 2 CH 2 CHC-t RNA

O

N H

CH O

N-Formylmethionine-tRNA fmet

Chain Initiation both tRNA met and tRNA fmet contain the triplet 3 -UAC-

5 this triplet base pairs with 5 -AUG- 3 in mRNA the 3 -UAC- 5 triplet on tRNA fmet recognizes the AUG

triplet (the start signal) when it occurs at the beginningof the mRNA sequence that directs polypeptidesynthesis

the 3 -UAC- 5 triplet on tRNA met recognizes the AUGtriplet when it is found in an internal position in themRNA sequence

the start signal is preceded by a Shine-Dalgarnopurine- rich leader segment, 5 -GGAGGU- 3, whichusually lies about 10 nucleotides upstream of the

AUG start signal and acts as a ribosomal binding site


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Chain Initiation The start of polypeptide synthesis requires an initiation

complex composed of mRNA 30S ribosomal subunit fmet-tRNA fmet GTP IF-3; facilitates binding of mRNA to the 30S subunit IF-2; binds GTP and aids in selection of fmet-tRNA fmet

IF-1; appears to facilitate binding of IF-3 and IF-2 50S ribosomal subunit

The binding of these units produces the 70S initiation complex

Chain Initiation

Figure 11.10Formation of aninitiation complex


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Chain Elongation

Requires 70S ribosome codons of mRNA aminoacyl-tRNAs elongation factors EF-Tu, EF-Ts, and EF-G GTP, and Mg 2+

See Figure 11.11 (next screen)

Chain Elongation Step 1

an aminoacyl-tRNA is bound to the A site the P site is already occupied

Step 2 EF-Tu is released in a reaction requiring EF-Ts

Step 3 the peptide bond is formed, the P site is uncharged

Step 4 the uncharged tRNA is released the peptidyl-tRNA is translocated to the P site EF-G and GTP are required the next aminoacyl-tRNA occupies the empty A site


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Chain Elongation

HH

O HO

H HOtRNA

An aminoacyl-tRNA

C= OH 2 N-CH

R

HH

O HO

H HOt RNA

f m e t

N-Formylmethionine-tRNA fmet

C= OH-C-N H-CH

CH 3 SCH 2 CH 2

O

HH

OH HO

H HO

t RNA f m e t

HH

O HO

H HO

t RNA

C= OH-C-N H-CH-C- NH- CH

CH 3 SCH 2 CH 2

O

R

O

peptidyl transferase

+

AdenineAdenine

Adenine Adenine

Chain Elongation

puromycin

HHO HO

H HO

C-CH-R

N H 3+

O

An aminoacyl-tRNA

N

N N

N

N H 2

t RNA- OPO- CH 2O -

HHN H HO

H HO

C-CH- CH 2

N H 3+

O

N

N N

N

N

HO - CH 2

CH 3H 3 C

OCH 3

Puromycin

O


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Amino Acid

A chain of nucleotidesmakes a codon (3letter word such as

ATT, GCA Each codon makes

an amino acid (20essential Amino

Acids)

Stop codons meanstranslation stops anda gene is complete

The Genetic Code

Features of the genetic code triplet: a sequence of three bases (a codon) is needed

to specify one amino acid nonoverlapping: no bases are shared between

consecutive codons

commaless: no intervening bases between codons degenerate: more than one triplet can code for the

same amino acid; Leu, Ser, and Arg, for example, areeach coded for by six triplets

universal: the same in viruses, prokaryotes, andeukaryotes; the only exceptions are some codons inmitochondria


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The Genetic Code

All 64 codons have been assigned 61 code for amino acids 3 (UAA, UAG, and UGA) serve as termination signals only Trp and Met have one codon each the third base is irrelevant for Leu, Val, Ser, Pro, Thr,

Ala, Gly, and Arg the second base is important for the type of amino

acid; for example, if the second base is U, the aminoacids coded for are hydrophobic

for the 15 amino acids coded for by 2, 3, or 4 triplets,it is only the third letter of the codon that varies. Gly,for example, is coded for by GGA, GGG, GGC, andGGU

1. Why is transcription necessary?Transcription makes messenger RNA (mRNA)to carry the code for proteins out of thenucleus to the ribosomes in the cytoplasm.

2. Describe transcription.RNA polymerase binds to DNA, separates thestrands, then uses one strand as a template toassemble mRNA.

3. Why is translation necessary?Translation assures that the right amino acidsare joined together by peptides to form thecorrect protein.


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4. Describe translation. The cell uses information from mRNA to

produce proteins. 5. What are the main differences between

DNA and RNA.DNA has deoxyribose, RNA has ribose;DNA has 2 strands, RNA has one strand;DNA has thymine, RNA has uracil.

6. Using the chart on Genetic Code, identify

the amino acids coded for by thesecodons: UGGCAGUGCtryptophan-glutamine-cysteine

AMAZING DNA FACTS DNA from a single human cell extends in

a single thread for almost 2 meters long!!! It contains information equal to some

600,000 printed pages of 500 wordseach!!!

(a l ibrary of abo ut 1 ,000 book s)

A string of codons codes for several aminoacids to form a gene

A gene can be as short as 50 nucleotidesand as long as 250 million.

Humans have over 3 billion nucleotides or 1billion codons

Each gene codes for a certain trait.


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In General, Genetic Engineering Techniques

Fall Into Two Classes

Identify a gene from anoth er species which controlsa trait of interest

Or modify an existing gene (create a new allele) DNA recombinant

Gene M anipulation

Introduces that gene into an organism Technique called transformation Forms tr ansgeni c organi sms

Gene I ntr oduction

Gene Manipulation StartsAt the DNA Level

The nucleus

contains DNA
http://en.wikipedia.org/wiki/Image:Gene.png


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An overview of how bacterial plasmids are used to clone genes

Using the Ti plasmid as a vector for genetic engineering in plants

Potential ApplicationsGenetically modify plants to...

produce vaccines in their fruit (e.g. polio vaccine)

be resistant to disease and pests

require less fertilizer, pesticides and herbicides

have a higher nutritional value


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Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissue Develop shoots Root the shoots

Field test the plants

Leaf, germinating seed, immature embryos

Tissue must be capable of developing into normal plants

Agrobacterium or gene gun (OTHER: Microinjection,Electroporation, Heat-shock, PEG

Multiple sites, multiple years

The Lab Steps


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Transformation cassettes are developed in the lab

They are then introduced into a plant

Two major delivery methods

Delivering the Geneto the Plant

Agrobacterium

Gene GunTissue culturerequired to generatetransgenic plants

Injection of DNA or a nucleus into CellPotential Applications1. Germ line Gene Therapy inject therapeutic gene into an egg cell (affects future

generations)

2. Somatic Gene Therapy Inject therapeutic gene into a somat ic ce l l , culture & reinsertinto an individual

3. Cloning inject nucleus into an enucleated egg, culture & implant into a surrogatemother.

Drawback: Inefficient means of gene transfer


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What Does It Mean: To Clone?

Clone: a collection of molecules or cells, all identical to anoriginal molecule or cell

To "clone a gene" is to make many copies of it - forexample, by replicating it in a culture of bacteria.

Cloned gene can be a normal copy of a gene (= wildtype).

Cloned gene can be an altered version of a gene (=mutant).

Recombinant DNA technology makes manipulating genespossible.

Genes Are Cloned Based On:

Similarity to known genes

H omology cloning (mouse clone used to obtain human gene)

Protein sequence

Complementary genetics (predicting gene sequencefrom protein)

Chromosomal location

M ap-based cloni ng (using genetic approach)


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2. Overview of various techniquesa. Use of Restriction Enzymes & DNA Ligase to

make recombinant DNA moleculesb. Use of Gel Electrophoresis...

To separate restriction fragments For DNA fingerprinting

c. PCR (Polymerase Chain Reaction)

Using a restriction enzyme and DNA

ligase to make recombinant DNA


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Gel Elec t rop ho res is

1. A method of separating mixtures of largemolecules (such as DNA fragments or proteins) onthe basis of molecular size and charge.

2. How its done An electric current is passed through a gel containing the

mixture Molecules travel through the medium at a different rates

according to size and electrical charge:Rate size and charge

Agarose and polyacrylamide gels are the mediacommonly used for electrophoresis of proteins andnucleic acids.

Gel electrophoresis of macromolecules


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Using restriction fragment patterns to distinguish DNA from different alleles

DNA fingerprints from a murder case

Whose blood is on the defendants clothing?


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PCR Polymerase Chain Reaction

A very quick, easy, automated method

used to make copies of a specific segmentof DNA Whats needed.

1. DNA primers that bracket the desiredsequence to be cloned

2. Heat-resistant DNA polymerase3. DNA nucleotides

4. Thermocycler

The polymerase chain

reaction (PCR)


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3. Strategies used to Genetically EngineerBacteria

An overview of how bacterial plasmids are used to clone genes

1. Isolate the gene of interest (e.g. insulin gene)2. Insert the gene of interest into a bacterial R-

plasmid R-plasmids are circular DNA molecules found in

some bacteria that provide resistance to up to 10different antibiotics

3. Place the transgenic plasmid into bacterialcells

Plasmid DNA reproduces each time the bacteria reproduce

4. Culture the bacteria and isolate the geneproduct (e.g. insulin)

3. Overview of how bacterial plasmids are used to clone genes

Figure 20.2


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Step 1. How to Isolate the Gene of Interest

Use Reverse Transc riptase to m ake the gene of Interest

Method #1 (see figure on next slide)1. Isolate mRNA for the gene product of interest (e.g.

Insulin mRNA)2. Use Reverse Transcriptase to produce cDNA

(complementary DNA)3. Use PCR to clone the cDNA3. Separate the synthetic gene of interest by

electrophoresis

Use of Reverse Transcriptase

to make complementary DNA(cDNA) of a eukaryotic gene


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Step 1. How to Isolate the Gene of Interest

Use Reverse Transc r iptase to m ake the gene ofInterest

Method #21. Determine the primary structure (i.e. the amino acid

sequence) of the protein of interest (e.g. insulin) with anautomated protein sequencer

2. Use table of codons to determine the mRNA sequence3. Synthesize the mRNA in the lab4. Use Reverse Transcriptase to produce cDNA and PCR

to clone the cDNA (as before)5. Separate the synthetic gene of interest by

electrophoresis

1. How to Isolate the Gene of InterestUse a labeled DNA Prob e to Isolate Gene of Interest (Southern Blot Method see

next slide) 1. Extract and purify DNA from cells2. Cut DNA with restriction enzyme (e.g. Eco R1)

Whats a restriction enzyme? ( fig. 20.3 ) Note: Must cut outside of gene w/o too much excess baggage

3. Separate DNA fragments by gel electrophoresis4. Transfer DNA from the fragile gel to a nylon sheet and heat to sep. strands ( fig.

20.10 )5. Hybridize gene of interest with a radio-labeled DNA* or mRNA* probe and expose

w/ film to locate gene How do these probes work? ( fig. 20.10 )

6. Use PCR to clone the isolated gene of interest.


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Figure 20.10 Restriction fragment analysis by Southern blotting

Steps 2 & 3. How to Insert the Gene of Interest into the R-

Plasmid See next 3 figures and animation Lyse bacteria with detergent to release the R-plasmid (e.g. ampicillin resistance

plasmid) Cut the plasmid with the same restriction enzyme used to isolate the gene of

interest3. Mix plasmid with gene of interest and join the two with DNA ligase

How does this work?4. Add the recombinant plasmid to a bacterial culture

Induce bacteria to take up plasmid (transformation)5. Grow bacteria on agar plate containing an antibiotic (e.g. ampicillin)6. Isolate those bacterial colonies that contain the recombinant plasmid How?

Only some of the bacteria take up a plasmid How do you know which ones did?Not all plasmids are recombinant plasmids How do you find those that are?

Only some of plasmids contain the gene of interest How do you identify these?


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Using Plasmids to Create Recombinant DNA

Using Plasmids to Create Recombinant DNA 1. Digest a plasmid vector with a restriction enzyme

(e.g. EcoRI) at a single site to produce two stickyends.

2. Digest human DNA with EcoRI to produce pieces withthe same sticky ends

Use Human DNA or cDNA copied from mRNA using reversetranscriptase from retroviruses.

3. Mix the two samples and allow to hybridize. Some plasmids will hybridize with pieces of human DNA at

the EcoRI site.

4. Use DNA ligase is used to covalently link thefragments.


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Insertion of Recombinant Plasmids into ProkaryoticCells

1. Only some of the bacteriatake up a plasmid How doyou know which ones did?

2. Not all plasmids arerecombinant plasmids How do you find those thatare?

3. Only some of plasmidscontain the gene ofinterest How do you

identify these?

Identification of cells containing plasmids

Cells containing plasmids contain theampicillin resistance gene

Grow cells on medium containing ampicillin How do you know which colonies contain

the gene of interest? Use a DNA probe (see fig. 20.5 )


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Figure 20.5 Using a DNA probe to

identify a cloned gene in apopulation of bacteria

Step 4. Culture Bacteria and Isolate Gene Product

Grow the recombinant bacteria in nutrientbroth and isolate/purify the gene productfrom the broth

Expensive to do, therefore mammals (e.g.cows and goats) are now being geneticallymodified to produce desired gene productsin their milk!!


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Human Gene Therapy using... a. Retroviruses

b. Adenovirusesc. Liposomes

d. Naked DNA

Use of a Retrovirusfor Gene Therapy

ApplicationsSomatic Gene Therapy to treat

Gaucher Disease

SCIDs Bubble Boy

(S evere Combined ImmuneDifficiency)


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Basic Strategies of Human Gene Therapy (1 of 2)

1. Isolate and then clone the normal allele by PCR 2. Insert normal allele into a disabled virus

Retroviruses and adenoviruses are the most common vectors Retroviruses are much more efficient at forming a provirus, but have a

greater chance of mutating to cause disease Adenoviruses are safer, but are relatively inefficient as a vector Liposomes (lipid spheres) are also used as vectors

e.g. Gene therapy for Cystic Fibrosis involves using an inhaler tobring liposomes containing the CFTR gene to the cells lining thelungs)

3. Infect host cells with recombinant virus

3. Infect host cells with recombinant virusa. Add recombinant virus directly to individual

e.g. Jesse Gelsinger Had Ornithine Transcarbamylase Deficiency; Causesbuild up of ammonia in liver cells since they cannotconvert the ammonia (toxic) produced by amino acidmetabolism to urea (less toxic)Died in Sept.99 due to a severe immune response to

the genetically modified adenovirus containing theOTC gene

b. Isolate host cells from body and then add recombinantvirus (e.g. blood stem cells in gene therapy for Gaucherdisease) Inject genetically engineered cells back into the body

Basic Strategies of Human Gene Therapy (2 of 2)


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Figure 20.6 Genomic libraries

Figure 20.11 Chromosome walking


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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 1)



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Figure 20.13 Alternative strategies for sequencing an entire genome

Table 20.1 Genome Sizes and Numbers of Genes


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Figure 20.14a DNA microarray assay for gene expression

Figure 20.14b DNA microarray assay for gene expression


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Application of Gene Manipulation

It is now routine to isolate genes

But the target gene must be carefully chosen

Target gene is chosen based on desired phenotype

Function:Glyphosate (RoundU p) resistance

EPSP synthase enzyme

I ncreased Vitami n A contentVitamin A biosynthetic pathway enzymes

The RoundUp Ready Story

Glyphosate is a broad-spectrum herbicide Active ingredient in RoundUp herbicide Kills all plants it come in contact with Inhibits a key enzyme ( EPSP synthase ) in an amino acid pathway

Plants die because they lack the key amino acids

A resistant EPSP synthase gene allows cropsto survive spraying


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+ Glyphosate

X

RoundUp Sensitive Plants

X

X

Shikimic acid + Phosphoenol pyruvate

3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)

PlantEPSP synthase

Aromaticamino acids

Without amino

acids, plant dies X

BacterialEPSP synthase

Shikimic acid + Phosphoenol pyruvate

3-enolpyruvyl shikimic acid-5-phosphate(EPSP)

Aromaticamino acids

RoundUp Resistant Plants

+ Glyphosate

With amino acids, plant lives

RoundUp has no effect;enzyme is resistant to herbicide


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The Golden Rice Solution

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

-carotene desaturase

Daffodil gene

Single bacterial gene;performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin APathway

is completeand functional

GoldenRice

Metabolic Pathways are Complexand Interrelated

Understanding pathwaysis critical to developing

new products


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Modifying Pathway ComponentsCan Produce New Products

Modified Lipids =New I ndustrial Oils

Turn On Vitamin Genes = Relieve Deficiency

Increase amino acids =I mproved Nutri tion

Use of a Retrovirusfor Gene Therapy

ApplicationsSomatic Gene Therapy to treat

Gaucher Disease

SCIDs Bubble Boy

(S evere Combined ImmuneDifficiency)

Documents

Lect 2- DNA and Molecular Genetic