Salty sweat due to altered salt secretion in sweat ducts Lung Pancreas Testis Infertility in males due to clogged sex ducts Mucus-clogged Airways; Severe Respiratory infections Symptoms of cystic fibrosis Cell lining ducts of 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

Parent molecule has 2 complementary strands of DNA 2.Enzymes initiate the breaking of the H-bonds, separating the double helix 3.Free nucleotides base – pair to parent strands (A-T, G-C) using DNA polymerase enzyme 4.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 Protein Cell membrane 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 is absent 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 Step 4 mRNA processing

Questions to answer 1.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 code Fig Codon = 3 letter section of mRNA that codes for one amino acid

Transfer RNA: tRNA tRNA Matches amino acids with codons in mRNA using anticodons Fig 10.15

Amino acid Codon on mRNA mRNA 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? MetLysPheGlyAla MetPheSerAla AACGGUUAUCCU Asn – Gly – Tyr – Pro Substitute C for U

Types of Mutations: Base Insertions and deletions Changes the reading frame of the genetic message MetLysLeuAlaHis (b) Nucleotide deletion mRNA Protein MetLysPheGlyAla

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