Chapter 21 DNA Biology and Technology Lecture … 105...Points to Ponder • What are three functions of DNA? • Review DNA and RNA structure. • What are the 3 types of RNA and

Human Biology

Sylvia S. Mader

Michael Windelspecht

Chapter 21

DNA Biology and Technology

Lecture Outline

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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Points to Ponder • What are three functions of DNA?

• Review DNA and RNA structure.

• What are the 3 types of RNA and what are their functions?

• Compare and contrast the structure and function of DNA and RNA.

• How does DNA replicate?

• Describe transcription and translation in detail.

• Describe the genetic code.

• Review protein structure and function.

• What are the 4 levels of regulating gene expression.

• What did we learn from the human genome project and where are we going from here?

• What is ex vivo and in vivo gene therapy?

• Define biotechnology, transgenic organisms, genetic engineering and recombinant DNA.

• What are some uses of transgenic bacteria, plants, and animals?

What must DNA do?

1. Replicate to be passed on to the next

generation

2. Store information

3. Undergo mutations to provide genetic diversity

21.1 DNA and RNA structure and function

DNA structure: A review

• Double-stranded helix

• Composed of repeating nucleotides (made of a

pentose sugar, phosphate, and a nitrogenous base)

• Sugar and phosphate make up the backbone while

the bases make up the “rungs” of the ladder

• Bases have complementary pairing with cytosine (C)

pairs with guanine (G) and adenine (A) pairs with

thymine (T)


DNA structure



T

S

P

T

S

P

S

P

P

P

S

P P

S

P

S

S

S

S

S

S

S S

b. Ladder structure a. Double helix c. One pair of bases

P

S

P

S

P

P

S S

S

S

P

P

P

O

C

C

C C

C

T A

G C

T A

C G

A T

T A

A

T

deoxyribose

5′ end

3′ end

3′ end

5′ end

5′ end 3′ end

5′

4′ 1′

3′ 2′

1′

2′ 3′

4′

5′

1′

2′ 3′

4′

5′

phosphate pyrimidine base purine base

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How does DNA replicate?

• The two strands unwind by

breaking the H bonds

• Complementary nucleotides

are added to each strand by

DNA polymerase

• Each new double-stranded

helix is made of one new

strand and one old strand

(semiconservative replication)

• The sequence of bases

makes each individual unique



A

A

A

T

G

G

G

C

C

DNA polymerase

new

strand

old

strand

DNA replication



A

A

A

T

G

G

G

C

C

DNA polymerase

new

strand

old

strand


Parental DNA molecule contains old

strands hydrogen-bonded by

complementary base pairing.

Region of replication. Parental DNA

is unwound and unzipped. New

nucleotides are pairing with those

in old strands.

Replication is complete. Each

double helix is composed of an old

(parental) strand and a new

(daughter) strand.













RNA structure and function

• Single-stranded

• Composed of repeating nucleotides

• Sugar-phosphate backbone

• Bases are A, C, G, and uracil (U)

• Three types of RNA

– Ribosomal (rRNA): joins with proteins to form ribosomes

– Messenger (mRNA): carries genetic information from DNA to

the ribosomes

– Transfer (tRNA): transfers amino acids to a ribosome where

they are added to a forming protein


RNA structure 21.1 DNA and RNA structure and function


S

S

S

S

G

U

A

C

G

U

A

C

ribose one nucleotide

P

P

P

P

Base is

uracil instead

of thymine.

Comparing DNA and RNA

• Similarities:

– Are nucleic acids

– Are made of

nucleotides

– Have sugar-phosphate

backbones

– Are found in the

nucleus

• Differences:

– DNA is double

stranded while RNA is

single stranded

– DNA has T while RNA

has U

– RNA is also found in

the cytoplasm as well

as the nucleus while

DNA is not


Proteins: A review

• Composed of subunits of amino acids

• Sequence of amino acids determines the shape of the

protein

• Synthesized at the ribosomes

• Important for diverse functions in the body including

hormones, enzymes and transport

• Can denature causing a loss of function

21.2 Gene expression

Proteins: A review of structure



C N

R

C

R

C N

C

R

C

N

C

R

N

C

R

N R

N

C

N R

hydrogen bond Secondary Structure:

Alpha helix or a pleated sheet

hydrogen bond

(beta) sheet=

pleated sheet (alpha) helix

Tertiary Structure:

final shape of polypeptide disulfide bond

CH

CH

CH

CH

CH

CH

CH

CH

peptide bond

H3N+

Primary Structure:

sequence of amino acids

amino acid

COO–

2 steps of gene expression

1. Transcription – DNA is read to make a mRNA in the nucleus of our cells

2. Translation – Reading the mRNA to make a protein in the cytoplasm



G

G

C

C

G

G

C

C

G

G

C

O O O

G

C

G

C

G

C

C

G

A

U

T

A

A

T

T

A

U

T

G

C

G

transcription

innucleus

DNA

double helix

DNA

mRNA

translation

at ribosome

polypeptide

tryptophan tyrosine arginine

codon 3 codon 2 codon 1

N C C N C C N C C

R1 R2 R3

The genetic code

• Made of 4 bases

• Bases act as a code for

amino acids in

translation

• Every 3 bases on the

mRNA is called a

codon that codes for a

particular amino acid in

translation


Second Base

arginine

CGG arginine

AGU serine

AGC serine

arginine

AGG arginine

GGU glycine

GGC glycine

glycine

GGG glycine

UGC cysteine

UGG tryptophan

CGU arginine

CGC arginine

stop

CAC histidine

glutamine

CAG glutamine

AAU asparagine

AAC asparagine

lysine

AAG lysine

GAU aspartate

GAC aspartate

glutamate

GAG glutamate

UUU phenylalanine

UUC phenylalanine

leucine

UUG leucine

CUU leucine

CUC leucine

leucine

CUG leucine

AUU isoleucine

AUC isoleucine

isoleucine

GUU valine

GUC valine

valine

GUG valine

UCC serine

serine

UCG serine

CCU proline

CCC proline

proline

CCG proline

ACU threonine

ACC threonine

threonine

ACG threonine

GCU alanine

GCC alanine

alanine

GCG alanine

UAC tyrosine

CAU histidine

stop

UAG stop

UCU serine

UAU tyrosine

UGU cysteine

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

U C A G

First

Base

Third

Base

AUG (start)

methionine

UGA UAA

CGA CUA

UUA UCA

CCA CAA

AUA ACA AAA AGA

GUA GCA GAA GGA


1. Transcription

• mRNA is made from a DNA template

• mRNA is processed before leaving the nucleus

• mRNA moves to the ribosomes to be read

• Every 3 bases on the mRNA is called a codon and codes for a particular amino acid in translation



Transcription is taking place

here—mRNA nucleotides are

being joined by the enzyme

RNA polymerase so that their

sequence is complementary

to a strand of DNA.

RNA

polymerase

DNA

template

strand

This mRNA transcript is

ready to be processed and

then it will move into the

cytoplasm.

Non-template

or “gene” strand

to mRNA processing

3′

5′

C

C G

T A

A T

T A

G C

C G

A T

C

A T

C G

T A

Processing of mRNA after transcription

Modifications of

mRNA:

• One end of the

RNA is capped

• Introns removed

• Poly-A tail is

added


e e e i i

e e e i i

e = exons

i = introns

DNA

DNA to be transcribed

transcription

e e e

(cut-out) (cut-out)

cap poly-A

tail

primary

RNA

mature

mRNA














2. Translation

3 steps: 1. Initiation: mRNA binds to the small ribosomal

subunit and causes 2 ribosomal units to associate

2. Elongation: polypeptide lengthens • tRNA picks up an amino acid

• tRNA has an anticodon that is complementary to the codon on the mRNA

• tRNA anticodon binds to the codon and drops off an amino acid to the growing polypeptide

3. Termination: a stop codon on the mRNA causes the ribosome to fall off the mRNA


Visualizing the 3 steps of translation



C

G

U A

U A

U

A

C A

U G G A C

C U G G A C U A A C C

C U G

t h r

2a. Elongation occurs in two stages.

first stage of elongation.

tRNA-polypeptide is at the P site

and a tRNA amino acid is at the

A site. The polypeptide will be

transferred to the tRNA-amino acid.

2b. Second stage of elongation. The

ribosome has moved to the right

and the tRNA polypeptide at the P site

is now longer by one amino acid. One

tRNA is outgoing and another tRNA

is incoming.

3. Termination. When the ribosome

reaches a stop codon, all participants

separate and the polypeptide is

released.

1. Initiation. The small ribosomal subunit,

them RNA, the first tRNA amino acid,

and the large ribosomal subunit

come together.

large ribosomal

subunit

free polypeptide

polypeptide bond

amino acid

polypeptide

small

ribosomal

subunit

met

met ser

ala

tryp

val

asp

thr

met

ser

ala

tryp

val

asp

met

ser

ala

tryp

val asp

A site P site













Overview of transcription and translation


3.mRNA moves into

cytoplasm and becomes

associated with ribosomes.

Translation 1. DNA in nucleus

serves as a template

for mRNA.

2. mRNA is processed

before leaving the nucleus.

primary

mRNA

mature

mRNA

DNA

TRANSCRIPTION

introns

exons


C C

G G

G

U

A

U U U

4. tRNAs with

anticodons carry

amino acids

to mRNA.

nuclear pore

mRNA

peptide

amino

acids

5. Anticodon–codon

complementary

base pairing occurs.

anticodon

codon ribosome

A A A

large and small

ribosomal subunits

tRNA

6. Polypeptide synthesis

takes place one amino

acid at a time.

Regulation of gene expression

4 levels: 1. Transcriptional control (nucleus):

• e.g., chromatin density and transcription factors

2. Posttranscriptional control (nucleus) • e.g., mRNA processing

3. Translational control (cytoplasm) • e.g., differential ability of mRNA to bind ribosomes

4. Posttranslational control (cytoplasm) • e.g., changes to the protein to make it functional


























DNA technology

1. Gene cloning through recombinant DNA

2. Polymerase chain reaction (PCR)

3. DNA fingerprinting

4. Biotechnology products from bacteria,

plants, and animals

21.3 DNA technology

DNA technology terms

• Genetic engineering – altering DNA in bacteria, viruses, plants, and animal cells through recombinant DNA techonology

• Recombinant DNA – contains DNA from 2 or more different sources

• Transgenic organisms – organisms that have a foreign gene inserted into them

• Biotechnology – using natural biological systems to create a product or to achieve an end desired by humans

21.3 DNA technology

21. 3 DNA technology

DNA Sequencing

• The order of nucleotides in a DNA sequence is

determined

• 1970s performed manually using dye-terminator

substances

• Now performed using dyes attached to

nucleotides and using a laser and computerized

machine to determine sequence

21. 3 DNA technology

Automated DNA Sequencer and an

Electropheragram Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Small section of Arabidopsis genome (sequencer): © Lawrence Berkeley Nat’l Lab/Roy Kaltschmidt, photographer













Polymerase chain reaction (PCR)

• Used to clone small

pieces of DNA

• Important for

amplifying DNA for

analysis such as in

DNA fingerprinting

21.3 DNA technology


PCR

cycles

first

second

third

fourth

fifth

and so forth

old new

new old

new new old old













DNA fingerprinting

• Fragments are

separated by their

charge/size ratios

• Results in a distinctive

pattern for each

individual

• Often used for paternity

or to identify an

individual at a crime

scene or unknown body

remains

21.3 DNA technology


Collect

DNA

Use gel electrophoresis to identify criminals

marker Suspect B Suspect A

Crime

scene

suspect

B

suspect

A

crime scene

evidence

Perform

PCR on

repeats

16 r

ep

eats

12 r

ep

eats

12 r

ep

eats

12 r

ep

eats

16 r

ep

eats

12 r

ep

eats













Gene cloning

• Recombinant DNA – contains DNA from 2 or more different sources that allows genes to be copies

• An example using bacteria to clone the human insulin gene:

– Restriction enzyme – used to cut the vector (plasmid) and the human DNA with the insulin gene

– DNA ligase: seals together the insulin gene and the plasmid

– Bacterial cells uptake plasmid and the gene is copied and product can be made

21.3 DNA technology

Visualizing gene cloning 21.3 DNA technology


A

Bacteria produce a product.

bacterium

human DNA

human cell

insulin gene

recombinant DN

4a 4b

Host cell takes up

recombined plasmid.

Restriction enzyme

cleaves DNA.

DNA ligase seals

the insulin gene

into the plasmid.

Gene cloning occurs.

3

2

1

plasmid

(vector)

insulin

Biotechnology products: Transgenic bacteria

• Important uses: – Insulin

– Human growth

hormone (HGH)

– Clotting factor VIII

– Tissue plasminogen activator (t-PA)

– Hepatitis B vaccine

– Bioremediation – cleaning up the environment

such as oil degrading bacteria

21.3 DNA technology

Biotechnology products: Transgenic plants

• Important uses: – Produce human proteins in their seeds such as

hormones, clotting factors and antibodies

– Plants resistant to herbicides

– Plants resistant to insects

– Plants resistant to frost

• Corn, soybean, and cotton plants are commonly genetically altered

• In 2008: – 92% of the soybeans and 80% of the corn planted in

the United States had been genetically engineered.

21.3 DNA technology

Biotechnology products: Transgenic plants

21.3 DNA technology


Transgenic Crops of the Future

Improved Agricultural Traits

Disease-protected Wheat, corn, potatoes

Wheat, rice, sugar beets, canola Herbicide-resistant

Salt-tolerant

Drought-tolerant

Cold-tolerant

Improved yield

Modified wood pulp

Fatty acid/oil content

Protein/starch content

Amino acid content

a. Desirable traits

Corn, soybeans

Cereals, potatoes, soybeans, rice, corn

Corn, soybeans

Cereals, rice, sugarcane



Cereals, rice, corn, cotton

Trees

Improved Food Quality Traits

b. Salt-intolerant Salt-tolerant

b(both): Courtesy Eduardo Blumwald

Health focus: Ecological concern about

BT crops

• Resistance increasing in the target pest

• Exchange of genetic material between the

transgenic plant and a related species

• Concern about the impact of BT crops on

nontarget species

21.3 DNA technology

Biotechnology products: Transgenic

animals

• Gene is inserted into the egg that when fertilized will develop into a transgenic animal

• Current uses: – Gene pharming: production of pharmaceuticals in the

milk of farm animals

– Larger animals: includes fish, cows, pigs, rabbits, and sheep

– Mouse models: the use of mice for various gene studies

– Xenotransplantation: pigs can express human proteins on their organs making it easier to transplant them into humans

21.3 DNA technology

Production of a transgenic animal

21.3 DNA technology


human gene

for growth

hormone

human growth

hormone

development within a host goat

Transgenic goat produces

human growth hormone.

microinjection of human gene

milk

fusion of enucleated

eggs with 2n

transgenic nuclei donor of eggs

donor of egg

Cloned

transgenic

goats produce

human growth

hormone.

development within a host goat

milk

What did we learn from the human

genome project (HGP)?

• Humans consist of about 3 billion bases and 25,000 genes

• Human genome sequenced in 2003

• There are many polymorphisms or small regions of DNA that vary among individuals were identified

• Genome size is not correlated with the number of genes or complexity of the organisms

21.4 Genomics

What is the next step in the HGP?

• Functional genomics

• Understanding how the 25,000 genes function

• Understanding the function of gene deserts (25% of

DNA is comprised of these regions)

• Comparative genomics

• Help understand how species have evolved

• Comparing genomes may help identify base

sequences that cause human illness

• Help in our understanding of gene regulation

21.4 Genomics

New endeavors

• Proteomics – the study of the structure,

function, and interactions of cell proteins

• Can be difficult to study because:

• protein concentrations differ greatly between cells

• protein location, concentration interactions differ from

minute to minute

• understanding proteins may lead to the discovery of better

drugs

• Bioinformatics – the application of computer

technologies to study the genome

21.4 Genomics

How can we modify a person’s genome?

• Gene therapy - insertion of genetic material into human cells to treat a disorder

– Ex vivo therapy – cells are removed for a person, altered, and then returned to the patient

– In vivo therapy – a gene is directly inserted into an individual through a vector (e.g., viruses) or directly injected to replace mutated genes or to restore normal controls over gene activity

• Gene therapy has been most successful in treating cancer

21.4 Genomics

Ex vivo gene therapy

21.4 Genomics


1. Remove bone

marrow stem cells.

defective gene

4. Return genetically

engineered cells

to patient.

normal gene

viral recombinant DNA

3.Viral recombinant

DNA carries normal

gene into genome.

reverse transcription

viral recombinant RNA

2. Use retroviruses

to bring the normal

gene into the bone

marrow stem cells.

viral

recombinant

RNA

normal gene

retrovirus

Documents

Chapter 21 DNA Biology and Technology Lecture … 105...Points to Ponder • What are three functions of DNA? • Review DNA and RNA structure. • What are the 3 types of RNA and