Salty sweat due toaltered salt secretionin sweat ducts

Lung

Pancreas

Testis

Infertility inmales dueto cloggedsex ducts

Mucus-cloggedAirways; Severe Respiratory infections

Symptoms of cystic fibrosis

Cell lining ductsof the body

Cystic Fibrosis• A single faulty protein is connected to the symptoms• In 1989 the gene was mapped to chromosome #7

Our Goals

• To determine the connection between the symptoms associated with cystic fibrosis and DNA – How?

• Learn how DNA replicates• Understand the genetic code and how the instructions in

a gene are used to make a protein

Central Dogma of Biology

• Nucleotide: base + sugar + phosphate

DNA and RNA: Polymers of Nucleotides

Fig 10.2

A single strand of DNA

Sugar- phosphate backbone

T

A

C

G

DNA vs. RNA Nucleotides• Four nucleotides are found in DNA

– Differ in their nitrogenous bases• Adenine (A), Thymine (T), Cytosine (C), Guanine

(G)– Sugar:

• Four nucleotides are found in RNA– Uracil instead of Thymine

• Adenine (A), Uracil (U), Cytosine (C), Guanine (G)– Sugar:

DNA is like a rope ladder twisted into a spiral

Twist

DNA Structure• Consists of 2

strands joined together by weak hydrogen bonds

• Rungs of the ladder are hydrogen bonded N-bases

Fig 10.4

Base pairing in DNA:

Figure 10.5

What is a gene?

• The kind of proteins an organism makes helps to determine it’s phenotype

The order of bases in a gene determines......

• The sequence of amino acids in the protein it codes for, which determines......

• the organism's phenotype—the physical and biochemical characteristics of an organism.

DNA replication

Questions to answer

1. When during the cell cycle does it occur?

2. What do we start with and end with?

3. Where does it occur in a cell?

4. What’s needed for it to occur?

5. What is the sequence of events?

6. Why is it said to be semi-conservative?

7. What does proofreading mean?

8. What does the proofreading?

DNA Replication

Fig 10.6

1 2 3 4

1. Parent molecule has 2 complementary strands of DNA2. Enzymes initiate the breaking of the H-bonds, separating

the double helix3. Free nucleotides base – pair to parent strands (A-T, G-C)

using DNA polymerase enzyme4. Each “daughter” strand consists of one parental strand

and one new strand –

DNA can be damaged – eg. by ultraviolet light

• The enzymes (e.g. DNA polymerase) can repair the damage:

– Is the damage always repaired? Consequences?

The Flow of Genetic

Information: DNA to RNA to

Protein• Transcription:

• Translation: ribosomes translate mRNA into protein—a chain of amino acids

Fig 10.9

CF phenotype• Genes

determine which proteins a cell can make

• Proteins control phenotype

• e.g. CFTR Gene codes for CFTR protein

CFTR Protein: The cystic fibrosis transmembrane regulator protein

Chloride ions

CFTR ProteinCellmembrane

Cytoplasm of cell lining duct or lungs CFTR Protein

• Pumps chloride ions (salt) out of cells lining ducts of the lungs

• What are the consequences when CFTR doesn’t work?

Inside of duct or

Air sac in lungs

The order of bases in a gene determines the order of amino acids in the protein it codes for

Fig 10.10

1. Enzymes: catalysts for nearly all chemical reactions in cells; Determine what cells can make and digest

2. Structural components:

3. Receptors on cell surface

4. Hormones: e.g. insulin, growth hormone, prolactin

5. Transport: e.g. hemoglobin, spindle fibers

6. Immune system: antibodies

Why are proteins so important?

Questions to answer:

1. What do we start with and end with?

2. Where does transcription occur? When?

3. What is needed for transcription to occur?

4. What is the sequence of events?

Transcription: copying DNA into RNA

An RNA Nucleotide

Phosphate

Sugar:ribose

This oxygen isabsent in deoxyribose

Base (Uracil, U)

RNA

• Base – pairing:

• Single stranded

• Sugar = ribose (one more oxygen than deoxyribose)

Stages of Transcription

Fig 10.13b

Fig 10.13a

Transcription

1. Initiation - RNA polymerase enzyme binds to the promotor (section of DNA indicating “start of a gene”)

2. Elongation – RNA polymerase catalyzes base pairing on the template strand (U-A, G-C)

3. Termination – RNA polymerase reaches the “stop” sequence and the new mRNA is released.

4. mRNA processing – non-coding regions of the mRNA are removed and the mRNA leaves the nucleus.

Fig. 10.14 Step 4 mRNA processing

Questions to answer1. What do we start with and end with?

2. Where does translation occur?

3. What is needed for translation to occur?

4. What is the sequence of events?

5. What are the roles of mRNA, ribosomes, start codon, tRNA, anticodons, stop codon?

Translation: Ribosomes reading mRNA to produce a polypeptide

How do ribosomes read the code?

The genetic codeFig 10.11Codon = 3 letter section of mRNA that codes for one amino acid

Transfer RNA: tRNAtRNA

• Matches amino acids with codons in mRNA using anticodons

Fig 10.15

Amino acid

Codon on mRNAmRNA

A portion of an mRNA molecule attached to a tRNA

Each Codon specifies a specific tRNA—amino acid

complex

1. Initiation – mRNA start codon binds to tRNA anticodon; Ribosome binds to both

Stages of translation

2. Elongation

• tRNA brings specific AAs to the ribosome as mRNA passes through the ribosome (codon – anticodon recognition)

3. Termination – Ribosome reads an mRNA stop codon (no tRNA with anticodon). mRNA and protein detach from the ribosome

Explaining the symptoms of CF

1. Mucus build-up in the lungs

– Lung infections (e.g. pneumonia)

2. Male sterility (blocked vas deferens)

3. Salty sweat

4. Trouble digesting food (blocked pancreatic duct)

Explaining the symptoms of CF

• In CF, the faulty CFTR protein never makes it to cell membrane

Understanding Cystic Fibrosis at the Cellular Level

How does CFTR protein get from where it’s produced to its home in the cell membrane?

1. Where is the CFTR protein produced?

2. Where does it go for modification? How does it get there?

3. How does the modified CFTR protein get to the plasma membrane?

4. The defective CFTR protein is recognized at the ER as defective Where is the defective CFTR protein sent?

CF symptoms may be mild or severe

Several hundred different mutations are associated with CF

CFTR Gene

What’s a Mutation?• Any change in the nucleotide sequence of DNA• Types of Mutations

• Mutations may Result from: – Random errors in DNA replication– Viruses– Chemicals/toxins (cigarette smoke)– Radiation (e.g. U.V. light, X-rays)

F508 deletion: the most common cause of cystic fibrosis

Mutation responsible for Sickle Cell Anemia: nucleotide substitution

Glu Val

3 Types of Mutations: Base Substitutions, Insertions or Deletions

• Base substitutions – May result in changes in

the amino acid sequence in a protein, or

– May be silent (have no effect) – Why?

Met Lys Phe Gly Ala

Met Phe Ser Ala

AACGGUUAUCCU

Asn – Gly – Tyr – Pro

Substitute C for U

Types of Mutations: Base Insertions and deletions

• Changes the reading frame of the genetic message

Met Lys Leu Ala His

(b) Nucleotide deletion

mRNA

Protein Met Lys Phe Gly Ala

• Although mutations are often harmful

– They are the source of the rich diversity of genes in the living world

– They contribute to the process of evolution by natural selection