learning objectives schedule and...

1

Learning Goal and Scale

1

Learning Objectives: 1. Relate how Grif fith’s bacterial experiments showed that a hereditary factor

was inv olved in transformation.

2. Summarize how Av ery’s experiments led his group to conclude that DNA is responsible f or transformation in bacteria.

3. Describe how Hershey and Chase’s experiment led to the conclusion that DNA, not protein, is the hereditary molecule in viruses.

4. Ev aluate the contributions of Franklin and Wilkes in helping Watson and Crick discover DNA’s double helix structure.

5. Describe the 3 parts of a nucleotide.

6. Summarize the role of covalent and hydrogen bonds in the structure of DNA.

7. Relate the role of the base-pairing rules to the structure of DNA.

8. Summarize the process of DNA replication.

9. Identify the role of enzymes in the replication of DNA.

10. Describe how complementary base pairing guides DNA replication.

11. Compare the number of replication forks in prokaryotic and eukaryotic cells during DNA replication.

12. Describe how errors are corrected during DNA replication.

13. Outline the f low of genetic information in cells from DNA to protein.

14. Compare the structure of RNA with that of DNA 2

Learning Objectives15. Summarize the process of transcription.

16. Describe the importance of the genetic code.

17. Compare the role of mRNA, rRNA, and tRNA in translation.

18. Identify the importance of learning about the human genome.

19. Distinguish between chromosome and gene mutations.

20. Explain the significance of non-coding DNA to DNA identification.

21. Describe four steps commonly used in DNA identification.

22. Explain the use of restriction enzymes, cloning vectors, and probes

in making recombinant DNA.

23. Summarize several applications of DNA identification.

24. Summarize insights gained from the human genome project.

25. Summarize the use of genetic engineering in medicine and

agriculture

26. Summarize gene therapy the pros and cons.

27. Discuss environmental and ethical issues associated with genetic engineering

3

Schedule and Announcements

• Quiz Friday on History of DNA

• Test 1 Jan 23

4

DNA Replication and Biotechnology

Chapter 10, 13

Timeline of Molecular history• 1859 Charles Darwin – no clue about

genes• 1865 Gregor Mendel – lost until 1900

• 1869 Friedrich Mieshcer - DNA• 1900 Hugo de Vries – rediscovers

Mendel• 1902 Archibold Garrod – genetic

cause of a human disease

• 1902 Sutton and Boveri –chromosome theory

• 1920 T.H. Morgan – genes on chromosomes/ Sex-linkage

• 1913 A.H. Sturtevant – linkage map

• 1927 H.J. Muller – x-ray induced mutations

• 1928-F. Griffith- Transformation

• 1941 G. Beadle and E.L. Taum – one gene = one enzyme

• 1944 O.T. Avery, C. McLeod, Maclyn McCarty - proposed DNA = genes

• 1950 E, Chargaff – DNA base composition rules

• 1952-Hershey and Chase

• 1953 J.Watson, F. Crick, R.Franklin and M.Wilkins – DNA structure

• 1958 M. Messelson and F. Stahl –DNA replication

• 1961 S.Brenner, F.Jacob, M. Messelson – mRNA discovered

• 1966 M. Niernberg, G.Khoroana –Genetic code

• 1970 H. Smith – restriction enzymes• 1972 P. Berg – first recombinant DNA

• 1973 H. Boyer, S.Cohen – first “cloned DNA” fragment

• 1977 W.Gilbert, F.Sanger – DNA sequencing – first virus sequenced

• 1981- first transgenic mammals

• 1985 Alec Jeffreys – DNA fingerprinting

• 1985 Kary Mullis – PCR amplification

• 1990 J.Watson Human Genome Project

• 1993 Huntinington’s Disease Group –first identified genetic disease gene

• 1995 C.VentnerH.Smith – base sequences of H. influenzae and M. genitalium

• 1997 I. Wilmut – cloned Dolly the Sheep

• 2000 Alain Fischer - first successful gene therapy trial

• 2003 Complete human genome sequenced

2

7

The Genetic Material

• Frederick Griffith, 1928

• Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia

• There are 2 strains of Streptococcus:

–S strain is virulent

–R strain is nonvirulent

• Griffith infected mice with these strains hoping to understand the difference

between the strains

Figure 13.2

LivingS cells(control)

Mouse healthy

Results

Experiment

Mouse healthy Mouse dies

Living S cells

LivingR cells(control)

Heat-killedS cells(control)

Mixture ofheat-killedS cells andliving R cells

Mouse dies

9


• Griffith’s conclusion:

–Information specifying virulence passed from the dead S strain cells into the live

R strain cells

–Griffith called the transfer of this

information transformation

10


• Avery, MacLeod, & McCarty, 1944

• Repeated Griffith’s experiment using purified cell extracts and discovered:

–removal of all protein from the transforming material did not destroy its ability to transform R strain cells

– DNA-digesting enzymes destroyed all transforming ability

– the transforming material is DNA

11


• Hershey & Chase, 1952

– investigated bacteriophages: viruses that infect bacteria

–the bacteriophage was composed of only DNA and protein

–they wanted to determine which of these molecules is the genetic material that is

injected into the bacteria

12


• Bacteriophage DNA was labeled with radioactive phosphorus (32P)

• Bacteriophage protein was labeled with radioactive sulfur (35S)

• radioactive molecules were tracked

• only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage

• conclusion: DNA is the genetic material

3

Figure 13.4

Labeled phagesinfect cells.

Batch 1: Radioactiv e sulfur (35S) in phage protein

Experiment

Agitation frees outsidephage parts from cells.

Centrifuged cellsform a pellet.

Radioactiv ity(phage protein)found in liquid

Batch 2: Radioactiv e phosphorus (32P) in phage DNA

Radioactiv ity (phage DNA) found in pellet

Radioactiv eprotein

Radioactiv eDNA

Centrifuge

Centrifuge

Pellet

Pellet

1 2 3

4

414

DNA Structure

• Determining the 3-dimmensional structure of

DNA involved the work of a few scientists:

–Erwin Chargaff determined that

• amount of adenine = amount of thymine

• amount of cytosine = amount of guanine

• This is known as Chargaff’s Rules

15

DNA Structure

• Rosalind Franklin and Maurice Wilkins

–Franklin performed X-ray diffraction studies to identify the 3-D structure

–discovered that DNA is helical

–discovered that the molecule has a

diameter of 2nm and makes a complete turn of the helix every 3.4 nm

Figure 13.6

(b) Franklin’s X-ray diffraction

photograph of DNA

(a) Rosalind Franklin

Watson and Crick• Watson and Crick built models of a double

helix to conform to the X-ray measurements and the chemistry of DNA

• Franklin had concluded that there were

two outer sugar-phosphate backbones, with the nitrogenous bases paired in the

molecule’s interior

• Watson built a model in which the

backbones were antiparallel (their

subunits run in opposite directions)

Watson and Crick

• At first, Watson and Crick thought the

bases paired like with like (A with A, and so on), but such pairings did not result in a

uniform width

• Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with

the X-ray data

4

Figure 13.UN02

Purine purine: too wide

Pyrimidine pyrimidine: too narrow

Purine pyrimidine: widthconsistent with X-ray data

Watson and Crick

• Watson and Crick reasoned that the

pairing was more specific, dictated by the base structures

• They determined that adenine (A) paired

only with thymine (T), and guanine (G) paired only with cytosine (C)

• The Watson-Crick model explains Chargaff’s rules: in any organism the

amount of A = T, and the amount of G = C

Figure 13.8

Sugar

Sugar

Sugar

Sugar

Thymine (T)Adenine (A)

Cytosine (C)Guanine (G)

Many proteins work together in DNA

replication and repair

• The relationship between structure and

function is manifest in the double helix

• Watson and Crick noted that the specific

base pairing suggested a possible copying

mechanism for genetic material

Figure 13.9-3

(a) Parentalmolecule

(b) Separation of parentalstrands into templates

(c) Formation of newstrands complementaryto template strands

T A

C G

CG

TA

TATA

T A

C G

CG

TA

T A

C G

CG

TA

TA

T A

C G

CG

TA

TA

The Basic Principle: Base Pairing to a Template Strand

• Since the two strands of DNA are

complementary, each strand acts as a template for building a new strand in

replication

• In DNA replication, the parent molecule unwinds, and two new daughter strands are

built based on base-pairing rules

5

• Watson and Crick’s semiconservative

model of replication predicts that when a double helix replicates, each daughter

molecule will have one old strand (derived or

“conserved” from the parent molecule) and one newly made strand

• Competing models were the conservative

model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old

and new)

Figure 13.10

(a) Conservativemodel

(b) Semiconservativemodel

(c) Dispersivemodel

Parent cellFirst

replicationSecond

replication

• Experiments by Matthew Meselson and

Franklin Stahl supported the semiconservative model

Figure 13.11

Conserv ativ emodel

Semiconserv ativemodel

Dispersiv emodel

Predictions: First replication Second replication

DNA samplecentrifugedafter firstreplication

DNA samplecentrifugedafter secondreplication

Bacteriacultured inmediumwith 15N(heav yisotope)

Bacteriatransferredto mediumwith 14N(lighterisotope)

Less dense

More dense

Experiment

Results

Conclusion

1

3

2

4

Getting Started

• Replication begins at particular sites called

origins of replication, where the two DNA strands are separated, opening up a

replication “bubble”

• At each end of a bubble is a replication fork, a

Y-shaped region where the parental strands

of DNA are being unwound

Figure 13.12

Single-strand bindingproteins

Helicase

Topoisomerase

Primase

Replicationfork

5

5

5

3

3

3

RNAprimer

6

• Helicases are enzymes that untwist the

double helix at the replication forks

• Single-strand binding proteins bind to and

stabilize single-stranded DNA

• Topoisomerase relieves the strain caused by

tight twisting ahead of the replication fork by

breaking, swiveling, and rejoining DNA strands

Figure 13.13

Double-strandedDNAmolecule

Twodaughter DNAmolecules

Replicationbubble

Replicationfork

Daughter(new) strand

Parental(template) strandOrigin of

replicationDouble-strandedDNA molecule

Two daughter DNA molecules

Bubble Replication fork

Daughter (new)strand

Parental (template)strand

Origin ofreplication

(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryoticcell

0.2

5

m

0.5

m

• DNA polymerases cannot initiate synthesis of

a polynucleotide; they can only add nucleotides to an already existing chain base-

paired with the template

• The initial nucleotide strand is a short RNA primer

Synthesizing a New DNA Strand

• The enzyme, primase, starts an RNA chain

from a single RNA nucleotide and adds RNA nucleotides one at a time using the parental

DNA as a template

• The primer is short (5–10 nucleotides long)

• The new DNA strand will start from the 3 end

of the RNA primer

• Enzymes called DNA polymerases catalyze

the elongation of new DNA at a replication fork

• Most DNA polymerases require a primer and

a DNA template strand

• The rate of elongation is about 500

nucleotides per second in bacteria and 50 per second in human cells

• Each nucleotide that is added to a growing

DNA consists of a sugar attached to a base and three phosphate groups

• dATP is used to make DNA and is similar to

the ATP of energy metabolism

• The difference is in the sugars: dATP has

deoxyribose, while ATP has ribose

• As each monomer nucleotide joins the DNA

strand, it loses two phosphate groups as a molecule of pyrophosphate

7

Figure 13.14

Pyro-

phosphate

New strand

Phosphate

Nucleotide

5 3

Template strand

Sugar

Base

5

3

5

3

5 3

DNA

poly-

merase

T

A T

C G

A

CG

C

P

P iP

i2

A T

C G

A

CG

C

Antiparallel Elongation

• The antiparallel structure of the double helix

affects replication

• DNA polymerases add nucleotides only to the

free

3end of a growing strand; therefore, a new DNA strand can elongate only in the

5to 3direction

• Along one template strand of DNA, the DNA

polymerase synthesizes a leading strand continuously, moving toward the replication

fork

• To elongate the other new strand, called the

lagging strand, DNA polymerase must work in

the direction away from the replication fork

• The lagging strand is synthesized as a series of segments called Okazaki fragments

• After formation of Okazaki fragments, DNA

polymerase I removes the RNA primers and replaces the nucleotides with DNA

• The remaining gaps are joined together by

DNA ligase

Figure 13.16

53

5

3

Origin of replicationLagging strand Lagging

strand

Ov erall directionsof replication

Leadingstrand

Leadingstrand

Ov erv iew

Primase makesRNA primer.

RNA primerfor fragment 1

Templatestrand

Okazakifragment 1

DNA pol IIImakes Okazakifragment 1.

DNA pol III

detaches.

53

5

3

5

3

5

3

RNA primerfor fragment 2

Okazakifragment 2 DNA pol III

makes Okazakifragment 2.

Ov erall direction of replication

DNA pol Ireplaces RNAwith DNA.

DNA ligase formsbonds betweenDNA fragments.

5

3

5

3

5

3

5

3

5

3

5

3

1

2

3

4

5

6

8

Proofreading and Repairing DNA

• DNA polymerases proofread newly made

DNA, replacing any incorrect nucleotides

• In mismatch repair of DNA, other enzymes

correct errors in base pairing

• A hereditary defect in one such enzyme is

associated with a form of colon cancer

• This defect allows cancer-causing errors to

accumulate in DNA faster than normal

• DNA can be damaged by exposure to

harmful chemical or physical agents such as cigarette smoke and X-rays; it can also

undergo spontaneous changes

• In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of

DNA

Figure 13.19-3

3

5

Nuclease

3

5

3

5

DNApolymerase

3

5

3

5 3

5

DNA ligase

3

5 3

5

• Eukaryotic chromosomal DNA molecules

have special nucleotide sequences at their ends called telomeres

• Telomeres do not prevent the shortening of

DNA molecules, but they do postpone it

• It has been proposed that the shortening of

telomeres is connected to aging

• If chromosomes of germ cells became

shorter in every cell cycle, essential genes would eventually be missing from the

gametes they produce

• An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells

• Telomerase is not active in most human

somatic cells

• However, it does show inappropriate activity

in some cancer cells

• Telomerase is currently under study as a

target for cancer therapies

9

Overview: The Flow of Genetic Information

• The information content of genes is in the

form of specific sequences of nucleotides in DNA

• The DNA inherited by an organism leads to

specific traits by dictating the synthesis of proteins

• Proteins are the links between genotype and phenotype

• Gene expression, the process by which DNA directs protein synthesis, includes two stages:

transcription and translation

Evidence from the Study of Metabolic Defects

• In 1902, British physician Archibald Garrod

first suggested that genes dictate phenotypes through enzymes that catalyze specific

chemical reactions

• He thought symptoms of an inherited disease reflect an inability to synthesize a certain

enzyme

• Cells synthesize and degrade molecules in a

series of steps, a metabolic pathway

Nutritional Mutants inNeurospora: Scientific Inquiry

• George Beadle and Edward Tatum disabled

genes in bread mold one by one and looked for phenotypic changes

• They studied the haploid bread mold because

it would be easier to detect recessive mutations

• They studied mutations that altered the ability of the fungus to grow on minimal medium

Figure 14.2

Neurosporacells

Each surv iv ingcell forms a colony of

geneticallyidentical cells.

No

growth

Growth

Mutant cells placedin a series of v ials,each containing

minimal mediumplus one additionalnutrient.

Surv iv ing cellstested for inabilityto grow on

minimal medium.

Indiv idual Neurosporacells placed on completegrowth medium. Growth

Cells subj ectedto X-rays.

Control: Wild-typecells in minimalmedium

2

1 3

4

5

• The researchers amassed a valuable

collection of Neurospora mutant strains, catalogued by their defects

• For example, one set of mutants all required

arginine for growth

• It was determined that different classes of

these mutants were blocked at a different step in the biochemical pathway for arginine

biosynthesis

Figure 14.3

Precursor

Gene A

Ornithine Citrulline Arginine

Gene B Gene C

Enzyme

A

Enzyme

B

Enzyme

C

10

The Products of Gene Expression: A Developing Story

• Some proteins are not enzymes, so

researchers later revised the one gene–one enzyme hypothesis: one gene–one protein

• Many proteins are composed of several

polypeptides, each of which has its own gene

• Therefore, Beadle and Tatum’s hypothesis is

now restated as the one gene–one polypeptide hypothesis

• It is common to refer to gene products as proteins rather than polypeptides

Basic Principles of Transcription and

Translation

• RNA is the bridge between DNA and protein

synthesis

• RNA is chemically similar to DNA, but RNA

has a ribose sugar and the base uracil (U)

rather than thymine (T)

• RNA is usually single-stranded

• Getting from DNA to protein requires two

stages: transcription and translation

• Transcription is the synthesis of RNA

using information in DNA

• Transcription produces messenger RNA

(mRNA)

• Translation is the synthesis of a

polypeptide, using information in the

mRNA

• Ribosomes are the sites of translation

• A primary transcript is the initial RNA

transcript from any gene prior to processing

• The central dogma is the concept that cells are

governed by a cellular chain of command

Figure 14.UN01

DNA RNA Protein

The Genetic Code

• How are the instructions for assembling amino

acids into proteins encoded into DNA?

• There are 20 amino acids, but there are only

four nucleotide bases in DNA

• How many nucleotides correspond to an

amino acid?

11

Codons: Triplets of Nucleotides

• The flow of information from gene to protein

is based on a triplet code: a series of nonoverlapping, three-nucleotide words

• The words of a gene are transcribed into

complementary nonoverlapping three-nucleotide words of mRNA

• These words are then translated into a chain of amino acids, forming a polypeptide

Figure 14.5

DNA

template

strand

Protein

mRNA

3

Trp

TRANSCRIPTION

TRANSLATION

Amino acid

Codon

5

35

3

5

Phe Gly Ser

GU G U UU G G UC C A

CA C A AA C C AG G T

GT G T TT G G TC C A

• During transcription, one of the two DNA

strands, called the template strand, provides a template for ordering the sequence of

complementary nucleotides in an RNA

transcript

• The template strand is always the same strand

for any given gene

• During translation, the mRNA base triplets,

called codons, are read in the 5 to 3direction

• Each codon specifies the amino acid (one

of 20) to be placed at the corresponding position along a polypeptide

Cracking the Code

• All 64 codons were deciphered by the mid-

1960s

• Of the 64 triplets, 61 code for amino acids; 3

triplets are “stop” signals to end translation

• The genetic code is redundant: more than

one codon may specify a particular amino

acid

• But it is not ambiguous: no codon specifies

more than one amino acid

• Codons must be read in the correct reading

frame (correct groupings) in order for the specified polypeptide to be produced

• Codons are read one at a time in a

nonoverlapping fashion

12

Figure 14.6

UUU

Second mRNA base

UUC

UUA

UUG

UCU

UCC

UCA

UCG

UAU

UAC

UAA

UAG

UGU

UGC

UGA

UGG

CUU

CUC

CUA

CUG

CCU

CCC

CCA

CCG

CAU

CAC

CAA

CAG

CGU

CGC

CGA

CGG

AUU

AUC

AUA

AUG

ACU

ACC

ACA

ACG

AAU

AAC

AAA

AAG

AGU

AGC

AGA

AGG

GUU

GUC

GUA

GUG

GCU

GCC

GCA

GCG

GAU

GAC

GAA

GAG

GGU

GGC

GGA

GGG

Fir

st m

RN

A b

as

e (

5e

nd

of

co

do

n)

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

U C A G

Phe

Leu

Ser

Tyr Cys

Trp

Met orstart

Stop

Stop Stop

Arg

Gln

His

ProLeu

Val Ala

Asp

Glu

Gly

IIeThr

Lys

Asn

Arg

Ser

Th

ird

mR

NA

ba

se

(3e

nd

of

cod

on

)

Evolution of the Genetic Code

• The genetic code is nearly universal, shared

by the simplest bacteria and the most complex animals

• Genes can be transcribed and translated after

being transplanted from one species to another

Figure 14.7

(a) Tobacco plant expressinga firefly gene

(b) Pig expressing a jellyfishgene

Transcription is the DNA-directed synthesis of RNA: a closer look

• Transcription is the first stage of gene

expression

Molecular Components of Transcription

• RNA synthesis is catalyzed by RNA

polymerase, which pries the DNA strands apart and joins together the RNA

nucleotides

• RNA polymerases assemble polynucleotides in the 5 to 3 direction

• However, RNA polymerases can start a chain without a primer

Figure 14.8-3Transcription unit

RNA polymerase

Promoter

Template strand of DNA

Start point

Termination

Completed RNA transcript

RNA transcript

UnwoundDNA

RewoundDNA

RNA transcript

Direction oftranscription(“downstream”)

Initiation

Elongation

35

35

35

3

5

35

35

35

35

35

35

3

2

1

13

Mutations of one or a few nucleotides can affect protein

structure and function• Mutations are changes in the genetic

material of a cell or virus

• Point mutations are chemical changes in just

one or a few nucleotide pairs of a gene

• The change of a single nucleotide in a DNA

template strand can lead to the production of

an abnormal protein

Figure 14.25

AG G

Wild-type hemoglobin

mRNA

5

3

mRNA

Wild-type hemoglobin DNA

5

35

3

TC C

TG G

AC C 5

3

AG G5 53 UG G 3

Normal hemoglobin

Sickle-cell hemoglobin

Mutant hemoglobin DNA

Sickle-cell hemoglobin

ValGlu

Types of Small-Scale Mutations

• Point mutations within a gene can be divided

into two general categories

–Nucleotide-pair substitutions

–One or more nucleotide-pair insertions or deletions

Figure 14.26

mRNA

DNA template strand

StopCarboxyl end

Protein

Amino end

Phe GlyMet Lys

A G3 T CC 5T T T TA A A AC C

5 3TA A A A AT T T TG G G G C

U5 3G CA U U G GGA A A AU U

Wild type

Phe GlyMet Lys

A A3 T CC 5T T T TA A A AC C

5 3TA A A A AT T T TG G G G T

U5 3G UA U U G GGA A A AU U

Phe SerMet Lys

A G3 T CC 5T T T TA A A AC T

5 3TA A A A AT T T TG G A G C

U5 3G CA U U A GGA A A AU U

Met

A C3 T CT 5A T A TC A A GC A

5 3TA T A T AG T T CG A T G G

U5 3G GA G U U GAU A U AU U

Leu AlaMet Lys

A A3 T GC 5T T T

A

A A C TC C

5 3TA A A AGT T AG G G C T

U5 3G UA U G G GGA A A

U

U A

GlyMet Phe

A T3 T TA 5A A

T T

C C G

C

C A

5 3TA T T

A

A

G G C

T

G T T A A

U5 3G AA G C U AUU U

A G

G

A

Met

A G3 T CC 5A T T TA A A AC C

5 3TA T A A AT T T TG G G G C

U5 3G UA U U G GGU A A AU U

Stop Stop

Stop

Stop

Stop

A instead of G

Silent (no effect on amino acid sequence)

(a) Nucleotide-pair substitution

U instead of C

T instead of C

Missense

A instead of G

A instead of T

Nonsense

U instead of A

Extra A

Frameshift causing immediate nonsense(1 nucleotide-pair insertion)

(b) Nucleotide-pair insertion or deletion

Extra U

Frameshift causing extensive missense(1 nucleotide-pair deletion)

missing

missing

missing

missing

No frameshift, but one amino acid missing(3 nucleotide-pair deletion)

Substitutions

• A nucleotide-pair substitution replaces one

nucleotide and its partner with another pair of nucleotides

• Silent mutations have no effect on the

amino acid produced by a codon because of redundancy in the genetic code

• Missense mutations still code for an amino

acid, but not the correct amino acid

• Substitution mutations are usually missense

mutations

• Nonsense mutations change an amino acid

codon into a stop codon, nearly always

leading to a nonfunctional protein

14

Insertions and Deletions

• Insertions and deletions are additions or

losses of nucleotide pairs in a gene

• These mutations have a disastrous effect on

the resulting protein more often than

substitutions do

• Insertion or deletion of nucleotides may alter

the reading frame of the genetic message, producing a frameshift mutation

Mutagens

• Spontaneous mutations can occur during DNA

replication, recombination, or repair

• Mutagens are physical or chemical agents

that can cause mutations

• Researchers have developed methods to test

the mutagenic activity of chemicals

• Most cancer-causing chemicals (carcinogens)

are mutagenic, and the converse is also true

Understanding DNA structure and replication makes genetic engineering possible

• Complementary base pairing of DNA is the

basis for nucleic acid hybridization, the base pairing of one strand of a nucleic acid to

another, complementary sequence

• Nucleic acid hybridization forms the foundation of virtually every technique used in

genetic engineering, the direct manipulation

of genes for practical purposes

DNA Cloning: Making Multiple Copies of a Gene or

Other DNA Segment

• To work directly with specific genes, scientists

prepare well-defined segments of DNA in identical copies, a process called DNA cloning

• Most methods for cloning pieces of DNA in

the laboratory share general features

• Many bacteria contain plasmids, small

circular DNA molecules that replicate separately from the bacterial chromosome

• To clone pieces of DNA, researchers first

obtain a plasmid and insert DNA from another source (“foreign DNA”) into it

• The resulting plasmid is called recombinant DNA

Figure 13.22

Copies of gene

Recombinantbacterium

Gene ofinterest

Gene used to alter bacteriafor cleaning up toxic waste

PlasmidBacterialchromosome

Gene for pest resistanceinserted into plants

Protein dissolv es blood clotsin heart attack therapy

RecombinantDNA (plasmid)

Bacterium Gene insertedinto plasmid

Plasmid put intobacterial cell

Cell containing geneof interest

DNA of chromosome(“foreign” DNA)

Gene of interest

Protein expressedfrom gene of interest

Human growth hormonetreats stunted growth

Protein harv ested

Host cell grown in culture to form a clone ofcells containing the “cloned” gene of interest

Basicresearchand v ariousapplications

1

2

3

4

15

• The production of multiple copies of a single

gene is called gene cloning

• Gene cloning is useful to make many copies

of a gene and to produce a protein product

• The ability to amplify many copies of a gene

is crucial for applications involving a single

gene

Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes cut DNA

molecules at specific DNA sequences called restriction sites

• A restriction enzyme usually makes many

cuts, yielding restriction fragments

Figure 13.23-3

Restriction enzyme cutsthe sugar-phosphatebackbones.

3

5

DNA3

5

DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

DNA ligaseseals the strands.

Sticky end

One possible combination

Recombinant DNA molecule

3

53

5 3

53

5

3

53

5

35

3

53

5 3

5

35

3

5

3

5 3

5

G

GGCCA

TTA

ATTA

GGCCA

TTA

ATTA

1

2

3

Restriction site

GGCCA

TTA

ATTA

GGC

• To see the fragments produced by cutting

DNA molecules with restriction enzymes, researchers use gel electrophoresis

• This technique separates a mixture of nucleic

acid fragments based on length

Figure 13.24

Mixture ofDNA mol-ecules ofdifferentsizes

Cathode

Restriction fragments

Anode

Wells

Gel

Powersource

(a) Negativ ely charged DNA molecules will movetoward the positiv e electrode.

(b) Shorter molecules are impeded less thanlonger ones, so they mov e faster through the gel. 90

DNA Analysis

• DNA fingerprinting

–An identification technique used to detect differences in the DNA of individuals

–Makes use of a variety of molecular procedures, including RFLP analysis

–First used in a US criminal trial in 1987

• Tommie Lee Andrews was found guilty

of rape

16

91

DNA AnalysisAmplifying DNA in Vitro: The Polymerase Chain Reaction

(PCR) and Its Use in Cloning

• The polymerase chain reaction, PCR, can

produce many copies of a specific target segment of DNA

–Kary Mullis (nobel prize for chemistry 1993)

• A three-step cycle brings about a chain

reaction that produces an exponentially

growing population of identical DNA molecules

• The key to PCR is an unusual, heat-stable

DNA polymerase called Taq polymerase.

Figure 13.25

3

5

Cycle 1yields 2 molecules

Genomic DNA

Denaturation

Target sequence

3

5

3

5

3

5

Primers

New nucleotides

Annealing

Extension

Cycle 2yields 4 molecules

Cycle 3yields 8 molecules;

2 molecules(in white boxes)

match target sequence

Technique

1

2

3

• PCR amplification alone cannot substitute for

gene cloning in cells

• Instead, PCR is used to provide the specific

DNA fragment to be cloned

• PCR primers are synthesized to include a

restriction site that matches the site in the

cloning vector

• The fragment and vector are cut and ligated

together

95

Medical Applications• Human proteins

– Medically important proteins can be produced in bacteria

• Human insulin

• Interf eron

• Atrial peptides

• Tissue plasminogen activator

• Human growth hormone

• Organs for Transplant (Xenotransplantation)

– Use of animal organs instead of human organs in transplant patients

– What animals would be good candidates?

• Tissue Engineering

– Bioartif icial organs-hybrids created from a combination of living cells and

biodegradable poly mers

– A. Atala and Harv ard medical team produced a working urinary bladder

96

Medical Applications

• Vaccines

–Subunit vaccines: Genes encoding a part of the protein coat are spliced into a

fragment of the vaccinia (cowpox)

genome

–DNA vaccines: Depend on the cellular

immune response (not antibodies)

17

97


98


• Gene therapy

– Adding a functional copy of a gene to correct a hereditary disorder

• Ex Vivo (outside the body) SCID patients

• In Vivo (inside the body) cystic fibrosis

• Sev ere combined immunodeficiency disease (SCID) i llustrates both

the potential and the problems

– Children lack the enzyme ADA (adenosine deaminase)

• Involved in the development of T and B cells

• Bone marrow stem cells

– Successful at first, but then patients developed a rare leukemia

99

Agricultural Applications

• Ti (tumor-inducing) plasmid is the most

used vector for plant genetic engineering

–Obtained from Agrobacterium

tumefaciens, which normally infects

broadleaf plants

–However, bacterium does not infect

cereals such as corn, rice and wheat

100


• Herbicide resistance

–Broadleaf plants have been

engineered to be

resistant to the herbicide

glyphosate

–This allows for no-till

planting– Roundup ready crops

101


• Pest resistance

– Insecticidal proteins have been transferred into crop plants to make them pest-resistant

• Bt toxin from Bacillus thuringiensis

• Bt cotton, corn, and peanuts

• Spider silk

– bulletproof vests, surgical thread, micro-conductors, optical f ibers and f ishing rods; even new types of clothing

• Golden rice

– Rice that has been genetically modif ied to produce b-carotene (provitamin A)

• Converted in the body to vitamin A

102

Agricultural Applications• Adoption of genetically modified (GM) crops has been resisted in some areas

because of questions about:

– Crop saf ety for human consumption

– Toxins produced

• Allergies

– Unapprov ed genetically modified corn was detected in Taco

Bell’s shells

» Massiv e recall of 2.8 million boxes of product

– Mov ement of genes into wild relatives

• Studies hav e shown that wind-carried pollen can cause transgenic crops to hy bridize with nearby weedy relatives

– Loss of biodiversity

– Creating “super-weeds”

• Pigweed

http://upload.wikimedia.org/wikipedia/commons/1/1d/Bt_plants.png

http://upload.wikimedia.org/wikipedia/commons/1/1d/Bt_plants.png

18

• Currently, up to 45 percent of U.S. corn is

genetically engineered as is 85 percent of soybeans. It has been estimated that 70-

75 percent of processed foods on

supermarket shelves--from soda to soup, crackers to condiments--contain

genetically engineered ingredients.

(Center for Food Safety)

103


• In January 2008, the

FDA approved the sale of meat and milk from cloned livestock,

despite the fact that Congress voted twice in 2007 to delay

FDA's decision on cloned animals until additional safety and

economic studies could be completed.

104Center f or Food Safety


105


• Biopharming

–Transgenic plants are used to produce pharmaceuticals

–Human serum albumin

–Recombinant subunit vaccines

• Against Norwalk and rabies viruses

–Recombinant monoclonal antibodies

• Against tooth decay-causing bacteria106

Agricultural Applications• Transgenic animal technology has not been

as successful as that in plants

–One interesting example is the

EnviroPig

• Engineered to carry the gene for the

enzyme phytase

• Breaks down phosphorus in feed

• Reduces excretion of harmful phosphates in the environment

• An attempt at Jurassic Park

–Cloning Woolly mammoth, saber-tooth

tiger

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