DNA, Genes, and Biotechnology

Chapter 22

Structure of the Hereditary Material

• Experiments in the 1950s showed that DNA is the hereditary material

• Scientists raced to determine the structure of DNA

• 1953 - Watson and Crick proposed that DNA is a double helix

Structure of Nucleotides in DNA

• Each nucleotide consists of– Deoxyribose (5-carbon sugar)

– Phosphate group

– A nitrogen-containing base

• Four bases– Adenine, Guanine, Thymine, Cytosine

(A) (G) (T) (C)

Nucleotide Bases

phosphate group

deoxyribose

ADENINE (A)

THYMINE (T)

CYTOSINE (C)

GUANINE (G)

Watson-Crick Model

• DNA consists of two nucleotide strands

• Strands run in opposite directions

• Strands are held together by hydrogen bonds between bases

• A binds with T, C with G

• Molecule is a double helix

Structure of DNA

A gene is a sequence of nucleotides in a DNA molecule

DNA Structure Helps Explain How It Duplicates

• DNA is two nucleotide strands held together by hydrogen bonds

• Hydrogen bonds between two strands are easily broken

• Each single strand then serves as template for new strand

DNA Replication

newnew old old

• Each parent strand remains intact

• Every DNA molecule is half “old” and half “new”

Base-Pairing during

Replication

Each old strand serves as the template for complementary new strand

Errors in DNA Replication

• Mistakes can occur during replication

• Enzymes may detect and correct the problem, restoring the proper DNA sequence

• When the error is not corrected, the result is a mutation

Gene Mutations

Base-Pair Substitutions

Insertions

Deletions

Effect of Base-Pair Substitution

original base triplet in a DNA strand

As DNA is replicated, proofreadingenzymes detect the mistake andmake a substitution for it:

a base substitution within the triplet (red)

One DNA molecule carries the original, unmutated sequence

The other DNAmolecule carries a gene mutation

POSSIBLE OUTCOMES:

OR

Frameshift Mutations

• Insertion– Extra base added into gene region

• Deletion– Base removed from gene region

• Both shift the reading frame

• Result in many wrong amino acids

Frameshift Mutation

ARGININE GLYCINE TYROSINE TRYPTOPHAN ASPARAGINE

ARGININE GLYCINE LEUCINE GLUTAMATELEUCINE

mRNA

PARENTAL DNA

amino acid sequence

altered mRNA

BASE INSERTION

altered amino-acid sequence

Transposable Elements

• DNA segments that move spontaneously about the genome

• When they insert into a gene region, they usually inactivate that gene

• Neurofibromatosis

DNA to RNA to Proteins

transcriptionDNA RNA

translation protein

Three Classes of RNAs

• Messenger RNA– Carries protein-building instruction

• Ribosomal RNA– Major component of ribosomes

• Transfer RNA– Delivers amino acids to ribosomes

Base-Pairing during Transcription

• A new RNA strand can be put together on a DNA region

DNA

DNA DNA

RNAG C A T

C G T A

G C A U

C G T A

base-pairing in DNA replication base-pairing in transcription

Promoter

• A base sequence in the DNA that signals the start of a gene

• For transcription to occur, RNA polymerase must first bind to a promoter

Gene Transcription

transcribed DNA winds up again

DNA to be transcribed unwinds

mRNAtranscript

RNA polymerase

Transcript Modificationunit of transcription in a DNA strand

exon intron

mature mRNA transcript

poly-A tail

5’

5’ 3’

3’

snipped out

snipped out

exon exonintron

cap

transcription into pre-mRNA

3’ 5’

Gene Regulation

• Most cells of your body carry the same genes

• Each uses only a tiny subset at any time, and some are never turned on

• Regulatory proteins can speed up or halt transcription

Genetic Code

• Set of 64 base triplets• Codons

– Nucleotide bases read in blocks of three

• 61 specify amino acids• 3 stop translation

Code Is Redundant• Twenty kinds of amino acids are specified by

61 codons

• Most amino acids can be specified by more

than one codon

• Six codons specify leucine

– UUA, UUG, CUU, CUC, CUA, CUG

tRNA Structurecodon in mRNA

anticodon in tRNA

amino acid OH

tRNA molecule’s attachment site for amino acid

Ribosomestunnel

small ribosomal subunit large ribosomal subunit intact ribosome

Three Stages of Translation

Initiation

Elongation

Termination

Initiation

• Initiator tRNA binds to small ribosomal subunit

• Small subunit/tRNA complex attaches to mRNA and moves along it to an AUG “start” codon

• Large ribosomal subunit joins complex

Binding Sites on Large Subunit

binding site for mRNA

P (first binding site for tRNA)

A (second binding site for tRNA)

Elongation

Termination

• A stop codon in the mRNA moves onto the ribosomal binding site

• No tRNA has a corresponding anticodon

• Proteins called release factors bind to the ribosome

• mRNA and polypeptide are released

Making Recombinant DNA

5’

3’

G

C T T A A

A A T T C

G

G A A T T C

C T T A A G3’

5’

one DNA fragment another DNA fragment

3’

5’

Restriction enzymes cut DNA into fragments

Fragments base-pair at sticky ends

Using Plasmids

• Plasmid is small circle of bacterial DNA

• Foreign DNA can be inserted into plasmid – Forms recombinant plasmids

– Plasmid is a cloning vector

– Can be used to deliver DNA into another cell

Using Plasmids

DNA fragments+enzymes

recombinantplasmids

host cells containing recombinant plasmids

Polymerase Chain Reaction

• Sequence to be copied is heated• Primers are added and bind to ends of

single strands• DNA polymerase uses free nucleotides

to create complementary strands• Doubles number of copies of DNA

PCR (1)double-stranded DNA to copy

DNA heated to 90°– 94°C

primers added to base-pair with ends

mixture cooled; base-pairing of primers and ends of DNA strands

DNA polymerasesassemble new DNA strands

PCR (2)mixture heated again; makes all DNA fragments unwind

mixture cooled; base-pairing between primers and ends of single DNA strands

DNA polymerase action again doubles number of identical DNA fragments

DNA Sequencing:Reaction Mixture

• Copies of DNA to be sequenced• Primer• DNA polymerase• Standard nucleotides• Modified nucleotides

Reactions Proceed

• Nucleotides are assembled to create complementary strands

• When a modified nucleotide is included, synthesis stops

• Result is millions of tagged copies of varying length

Recording the Sequence

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

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

T C C A T G

T C C A T

T C C A

T C C

T C

T

electrophoresisgel

one of the many fragments of DNA migratingthrough the gel

one of the DNA fragmentspassing through a laser beam after moving through the gel

T C C A T G G A C C A

•DNA is placed on gel•Fragments move off gel in size order; pass through laser beam•Color each fragment fluoresces is recorded on printout

The Human Genome Project

Goal - Map the entire human genome

• Initially thought by many to be a waste of resources

• Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes

• Sequencing was completed ahead of schedule in early 2001

Surprising Discoveries

• Coding regions (exons) make up only 1.5% of our DNA

• About half of remaining DNA is repeated segments

• Around 1.4 million SNPs in the human genome (single nucleotide polymorphisms)

Using Human Genes• Even with gene in hand it is difficult to

manipulate it to advantage• Viruses usually used to insert genes

into cultured human cells but procedure has problems

• Very difficult to get modified genes to work where they should

DNA Fingerprints

• Unique array of DNA fragments

• Inherited from parents in Mendelian fashion

• Even full siblings can be distinguished from one another by this technique

Tandem Repeats

• Short regions of DNA that differ substantially among people

• Many sites in genome where tandem repeats occur

• Each person carries a unique combination of repeat numbers

RFLPs

• Restriction fragment-length polymorphisms• DNA from areas with tandem repeats is cut

with restriction enzymes• Because of the variation in the amount of

repeated DNA, the restriction fragments vary in size

• Variation is detected by gel electrophoresis

Biotechnology Concerns

• Mutation of transgenic bacteria or viruses could yield new pathogens

• Bioengineered plants and animals could alter ecological balance

• Genetic screening could lead to discrimination

Engineered Bacteria

• Transgenic bacteria can be used to grow medically valuable proteins– Insulin, interferon, blood-clotting factors

– Vaccines

• Human gene is inserted into bacteria, which are then grown in huge vats

Engineered Plants and Animals

• Transgenic goats produce human tissue plasminogen activator (tPA)

• Chinese hamster ovary cells produce blood-clotting factor VIII

• Aspen plants produce less lignin and more cellulose

Jefferson’s Genes

• It was suspected that Jefferson had at least one child with his slave, Sally Hemings

• DNA evidence showed that a modern male descendant of Sally Hemings has a Y chromosome that had been passed down to him through the generations from Thomas Jefferson