Inquiry into LifeTwelfth Edition

Chapter 24

GCU

DNA, RNA

Lecture PowerPoint to accompany

Sylvia S. Mader

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1

24.1 DNA Structure and Replication

• Hershey-Chase Experiments (1952)

– Demonstrated that DNA is the genetic material that directs all cells

– DNA stands for Deoxyribonucleic Acid

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Hershey-Chase Experiments

http://highered.mcgraw-hill.com/olc/dl/120076/bio21.swf 3

24.1 DNA Structure and Replication

• Structure of DNA

– James Watson and Francis Crick determined

the structure of DNA in 1953

– DNA is a chain of nucleotides

– Each nucleotide is a complex of three subunits• Phosphoric acid (phosphate)

• A pentose sugar (deoxyribose)

• A nitrogen-containing base (adenine, guanine,

cytosine, thymine)

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24.1 DNA Structure and Replication

• Structure of DNA

– Four Possible Bases• Adenine (A) - a purine

• Guanine (G) - a purine

• Thymine (T) - a pyrimidine

• Cytosine (C) - a pyrimidine

– Complimentary Base Pairing• Adenine (A) always pairs with Thymine (T)

• Guanine (G) always pairs with Cytosine (C)

• http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation12.html

N H O CH3

NN

O

N

N

NN H

Sugar

Sugar

Adenine (A) Thymine (T)

N

N

N

NSugar

O H NH

NH

N OHH

N

Sugar

Guanine (G) Cytosine (C)

H

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Overview of DNA Structure

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24.1 DNA Structure and Replication

• Replication of DNA

– Semi-conservative replication

• Each daughter DNA molecule consists of one new chain of

nucleotides and one from the parent DNA molecule

– The two daughter DNA molecules will be identical to

the parent molecule

(a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

(b) The first step in replication is separation of the two DNA strands.

(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.

ACTAG

ACTAG

ACTAG

ACTAG

TGATC

TGATC

ACTAG

ACTA

G

TGATC

TGATC

TGATC

TGATC

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24.1 DNA Structure and Replication

• Replication of DNA– Before replication begins, the two strands of the parent molecule

are hydrogen-bonded together between the bases

– DNA helicase (enzyme) unwinds and “unzips” the double-

stranded DNA

– New complementary DNA nucleotides fit into place along divided

strands by complementary base pairing. These are positioned

and joined by DNA polymerase (enzyme)

– DNA ligase (enzyme) repairs any breaks in the sugar-

phosphate backbone

– The Two double helix molecules identical to each other and to

the original DNA molecule8

Overview of DNA Replication

http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation16.html

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Ladder Configuration and DNA Replication

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24.2 Gene Expression• Gene: A segment of DNA that specifies

the amino acid sequence of a polypeptide

• DNA does not directly control protein synthesis, instead its information is transcribed into RNA

• The “Central Dogma”:

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24.2 Gene Expression

• RNA (ribonucleic acid)

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24.2 Gene Expression

• Three Classes of RNA– Messenger RNA (mRNA)

• Takes a message from DNA to the ribosomes• strand

– Ribosomal RNA (rRNA)• Makes up ribosomes (along with proteins)• globular

– Transfer RNA (tRNA)• Transfers amino acids to ribosomes• Hairpin shape

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24.2 Gene Expression

• Gene Expression Requires Two Steps:• Transcription

– Is the synthesis of RNA under the direction of DNA– Produces messenger RNA (mRNA)

• Translation– Is the actual synthesis of a polypeptide, which occurs

under the direction of mRNA– Occurs on ribosomes

http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation1.html

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Ribonucleic Acid• Why would the cell want to have an intermediate

between DNA and the proteins it encodes? – The DNA can then stay pristine and protected,

away from the caustic chemistry of the cytoplasm.

– Gene information can be amplified by having many copies of an RNA made from one copy of DNA.

– Regulation of gene expression can be effected by having specific controls at each element of the pathway between DNA and proteins.

– The more elements there are in the pathway, the more opportunities there are to control it in different circumstances. 15

24.2 Gene Expression

• Transcription– During transcription, a segment of the DNA serves as a template

for the production of an RNA molecule

– Messenger RNA (mRNA)• RNA polymerase (enzyme) binds to a promoter (“start”

sequence)• DNA helix is opened so complementary base pairing can

occur• RNA polymerase joins new RNA nucleotides in a sequence

complementary to that on the DNA, in a 5’ to 3’ direction

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Transcription of DNA to form mRNA

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Messenger RNA

• mRNA - of the 64 possible 3-base combinations:– 61 code for the twenty different amino acids – 3 code for "stop"; i.e. chain termination

• Specific nucleotide sequences call for “start” of transcription (usually  AUG = methionine) = PROMOTOR sequence

• “stop” of mRNA synthesis = TERMINATION sequence (UAA, UGA, UAG)

• Finished mRNA strands are ~500-10,000 nucleotides long

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• During transcription– The gene determines the sequence of bases along

the length of an mRNA molecule

Figure 17.4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNA strand(template)

TRANSCRIPTION

mRNA

Protein

TRANSLATION

Amino acid

A C C A A A C C G A G T

U G G U U U G G C U C A

Trp Phe Gly Ser

Codon

3 5

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24.2 Processing of mRNA

• After Transcription

• Primary “Pre-”mRNA must be modified into

mature mRNA– Introns are intragene segments (often, junk)

– Exons are the portion of a gene that is expressed

• Intron sequences are removed, and a poly-A tail is

added– Ribozyme splices exon segments together

– http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation20.html

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mRNA Processing pre-RNA must be modified before translation

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The Functional and Evolutionary Importance of Introns

• The presence of introns– Allows for alternative RNA splicing– Animations of RNA processing:

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/bio25.swf::Processing%20of%20Gene%20Information%20-%20Prokaryotes%20versus%20Eukaryotes

– http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/bio30.swf::How%20Spliceosomes%20Process%20RNA

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24.2 Gene Expression

• Translation

– The Genetic Code• Triplet code: each 3-nucleotide unit of a mRNA molecule is

called a codon

• There are 64 different mRNA codons

– 61 code for particular amino acids

» Redundant code; some amino acids have numerous code

words

» Provides some protection against mutations

– 3 are stop codons signal polypeptide termination

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Messenger RNA Codons

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Overview of Gene Expression

Protein synthesishttp://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis

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24.2 Gene Expression• Transfer RNA

– tRNA transports amino acids to the

ribosomes (creates polypeptide chain)

– Single stranded nucleic acid that

correlates a specific nucleotide sequence

with a specific amino acid

– Amino acid binds to one end, the

opposite end has an anticodon

– the order of mRNA codons determines

the order in which tRNA brings in amino

acids

Protein synthesishttp://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis

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Transfer RNA: Amino Acid Carrier

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rRNA

– Ribosomal RNA is the most abundant type of RNA in cells

– Ribosomes: comprised of subunits 2/3 RNA, 1/3 protein

• Two populations of ribosomes are evident in cells, Free and bound• Free ribosomes in the cytosol initiate the synthesis of all proteins

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• The ribosome has three binding sites for tRNA– The P site– The A site– The E site

Figure 17.16b

E P A

P site (Peptidyl-tRNAbinding site)

E site (Exit site)

mRNAbinding site

A site (Aminoacyl-tRNA binding site)

Largesubunit

Smallsubunit

Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams.

(b)

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• Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look

Quicktime movie: http://carbon.cudenver.edu/~bstith/transla.MOV

Narrated animation: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis

Interactive practice: http://learn.genetics.utah.edu/content/begin/dna/transcribe/

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24.2 Gene Expression

• Ribosome and Ribosomal RNA– Ribosome has a binding site for mRNA and

for 2 tRNAs– Facilitates complementary base pairing– Ribosome moves along mRNA and new

tRNAs come in and line up in order– This brings amino acids in line in a specific

order to form a polypeptide– Several ribosomes may move along the

same mRNA• Multiple copies of a polypeptide may be made• The entire complex is called a polyribosome

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Polyribosomes• A number of ribosomes can translate a single

mRNA molecule simultaneously– Forming a polyribosome

Figure 17.20a, b

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start of mRNA(5 end)

End of mRNA(3 end)

Polyribosome

An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

(a)

Ribosomes

mRNA

This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

0.1 µm(b)

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Translation (Building a polypeptide) requires Three Steps:

– Initiation (requires energy)– Elongation (requires energy)– Termination

Amino end Growing polypeptide

Next amino acidto be added topolypeptide chain

tRNA

mRNA

Codons

3

5

Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site.

(c)

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Summary of Gene Expression

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24.2 Gene Expression

• Genes and Gene Mutations– A gene mutation is a change in the sequence of

bases within a gene.

– Gene mutations can lead to malfunctioning proteins in cells.

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24.2 Gene Expression

• Genes and Gene Mutations– Causes of Mutations

• Errors in replication– Rare– DNA polymerase “proofreads” new strands and errors

are cleaved out

• Mutagens– Environmental influences– Radiation, UV light, chemicals– Rate is still fairly low because DNA repair enzymes

monitor and repair DNA

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Transposons

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Transposons• “jumping genes”• Can move to new locations and disrupt gene sequences

Types of Gene Mutations

Point Mutations– The substitution of one nucleotide for another

• Missense mutations – a point mutation in which a single nucleotide is changed, resulting in a

codon that codes for a different amino acid– Missense mutations are responsible for about 75% of the mutations in the

p53 gene. Mutations of this gene are responsible for about 30-50% of cancers in humans

• Silent mutations• Nonsense mutations

– mutations that change an amino acid to a stop codon 38

Types of Gene Mutations

• Frameshift Mutations– One or more nucleotides

are inserted or deleted– Results in a polypeptide

that codes for the wrong sequence of amino acids

– Codons must be read in the correct reading frame for the specified polypeptide to be produced

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DNA Technology• In laboratory experiments

– Genes can be transcribed and translated after being transplanted from one species to another

• Called “Recombinant DNA” technology• Can be produced via “Genetic Engineering” (laboratory manipulation)

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Overview: Understanding and Manipulating Genomes

• One of the greatest achievements of modern science has been the sequencing of the human genome, which was largely completed by 2003

• DNA sequencing accomplishments– Have all depended on advances in DNA

technology, starting with the invention of methods for making recombinant DNA

– DNA sequencing animation

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DNA Cloning

• Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment

• To work directly with specific genes– Scientists have developed methods for

preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning

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24.3 DNA Technology

• The Cloning of a Gene

– Cloning: Production of many identical copies of

an organism through some

asexual means.

– Gene Cloning: The production of many identical copies of a single gene

– Two Ways to Clone a Gene:– Recombinant DNA

– Polymerase Chain Reaction

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Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes– Cut DNA molecules at

a limited number of specific DNA sequences, called restriction sites

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Restriction Enzymes and Sticky Ends

Step through animation of cut/splice using EcoRI

Narrated animation45

Cloning of a Human Gene / Recombinant DNA

– Restriction enzymes breaks open a plasmid vector at specific sequence of bases “sticky ends”

– Foreign DNA that is to be inserted is also cleaved with same restriction enzyme so ends match

– Foreign DNA is inserted into plasmid DNA and “sticky ends” pair up

– DNA ligase seals them together

– Narrated animation of “Cloning a Gene” 46

24.3 DNA Technology

• Polymerase Chain Reaction– Amplifies a targeted DNA sequence– Requires DNA polymerase, a set of primers, and a

supply of nucleotides• Primers are single stranded DNA sequences that start

replication process

– Amount of DNA doubles with each replication cycle– Process is now automated– Narrated animation– Step by step animation

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Polymerase Chain Reaction (PCR)

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24.3 DNA Technology• DNA Fingerprinting

– Permits identification of individuals and their relatives– Based on differences between sequences in

nucleotides between individuals– RFLPs : restriction fragment length polymorphisms– Narrated animation– Detection of the number of repeating segments (called

repeats) are present at specific locations in DNA• Different numbers in different people• PCR amplifies only particular portions of the DNA• Procedure is performed at several locations to identify

repeats

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DNA Fingerprints

DNA fragments (after digest with restriction enzymes) can be separated through gel ELECTROPHORESIS

See How:Step-by-step electrophoresis

Another walk-through explanation

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Forensic Evidence• DNA “fingerprints”

obtained by analysis of tissue or body fluids found at crime scenes– Can provide definitive

evidence that a suspect is guilty or, more specifically, not guilty

– Is a specific pattern of bands of RFLP markers on a gel

Defendant’sblood (D)

Blood fromdefendant’sclothes

Victim’sblood (V)

D Jeans shirt V

4 g 8 g

Figure 20.1751

DNA fingerprinting

Can also be used in establishing paternity

Figure: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Paternity_testing

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24.3 DNA Technology

• Biotechnology

– Biotechnology uses natural biological systems to create a product or to achieve a goal desired by humans.

– “Model Organisms” favored for genetics research

– Transgenic organisms have a foreign gene inserted into their DNA

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– Have been engineered to be pharmaceutical “factories”

Figure 20.18 “Pharm” Animals 54

Pharmaceutical Products

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24.3 DNA Technology

• Transgenic Bacteria

– Medical Uses: Production of Insulin, Human Growth

Hormone, Hepatitis B Vaccine

– Agricultural Uses: Bacteria that protects plants from freezing, bacteria that protect plant roots from insects

– Environmental: Bacteria that degrade oil (clean up after oil spills), bacteria that remove sulfur from coal

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Environmental Cleanup• Genetic engineering can be used to modify

the metabolism of microorganisms– So that they can be used to extract minerals

from the environment or degrade various types of potentially toxic waste materials

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24.3 DNA Technology

• Transgenic Plants

– Plants have been engineered to secrete a toxin that

kills insects (ex: Bt corn)

– Plants have been engineered to be resistant to

herbicides (ex: Roundup Ready)

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Agricultural Applications• DNA technology

– Is being used to improve agricultural productivity and food quality

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Genetic Engineering in Plants• Agricultural scientists

– Have already endowed a number of crop plants with genes for desirable traits

Bt corn (right) 60

24.3 DNA Technology

• Transgenic

Animals

– Fish, cows, pigs,

rabbits and sheep

have been

engineered to

produce human

growth hormone in

order to increase

size of the animals

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Animal Husbandry and “Pharm” Animals

• Transgenic animals– Contain genes from other organisms– Sometimes called “chimeras”– Fig 1. transgenic mouse lines expressing GFP known as “green

mice.”

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Animal Husbandry and “Pharm” Animals

• “Knockout” mice

Since cancer is a multistage process, it is obvious that transgenic or knock out animals, which have already undergone one step in the cancer process, may be a sensitive alternative for the standard bioassay.

A number of mice models have been developed: either possessing an inactivated tumor suppressor gene (p53), an activated oncogene (Tg.AC), over-expression of a (human) oncogene (rasH2) or being deficient in nucleotide excision repair (Xpa, de Vries et al., 1995).

These mice models have several advantages: • the number of animals needed for one study is 120 instead of 400-500• the duration of the study is 6-9 instead of 24 months leading to less distress of the animals• the transgenic mouse model is considered more discriminating hence improving the accuracy

and reliability of human carcinogen identification.

http://www.nca-nl.org/English/Newsletters/Nb13/nl13txt.html

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Safety and Ethical Questions Raised by DNA Technology

• The potential benefits of genetic engineering– Must be carefully weighed against the potential

hazards of creating products or developing procedures that are harmful to humans or the environment

• Today, much public concern about possible hazards

– Centers on genetically modified (GMOs) organisms used as food

– Gene “escape” 64

Transgenic Animals

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