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Gene Mutations. Mutation Vocabulary. Gene Mutation . Chromosomal Mutation. Duplication Deletion Translocation Inversion. Point Mutation Substitution Silent Missense Nonsense Frameshift Insertion Deletion. Chromosomal Mutations. How do genes control metabolism.
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Mutation Vocabulary Gene Mutation Chromosomal Mutation Duplication Deletion Translocation Inversion • Point Mutation • Substitution • Silent • Missense • Nonsense • Frameshift • Insertion • Deletion
How do genes control metabolism • In 1909, Archibald Garrod first proposed the relationship between genes and proteins. • “Genes dictate phenotypes through enzymes that catalyze specific chemical processes in the cell.” • Example: Alkaptonuria – Urine appears dark red because it contains alkapton, that darkens upon exposure to air. • Wild Type Individuals have an enzyme to break the chemical down. • Mutant Individuals do not produce the enzyme, thus unable to metabolize alkapton.
Beadle and Tatum One Gene – One Enzyme hypothesis : the Function of one gene is to dictate the production of a specific enzyme.
Transcription in Prokaryotes • RNA polymerase cannot initiate transcription on its own. A protein (sigma) must bind before transcription can begin. • RNA Polymerase + Sigma = HOLOENZYME • When a holoenzyme + DNA mix, the enzyme will attach to only specific regions on the DNA which they now refer to as PROMOTERS. Specifically positions 10 and 35 nucleotides upstream from the gene. • The sigma appeared to be responsible for guiding the RNA polymerase to specific locations. • The sigma will release once initiation has commenced.
Promoter Sequence • 20 – 25 base pairs long. • Similar segment of DNA had a series of bases identical or similar to TATAAT. • Referred to as the – 10 box.
Translation in Prokaryotes • A large and small subunit assemble onto the mRNA. The small subunit will attach to what is called the Shine-Dalgarno Sequence on the mRNA. • A 6 base sequence upstream 8 bases from the AUG start codon. The rRNA sequence within the small subunit will attach. • Shine-Dalgarno Sequence – AGGAGG • Anti Shine-Dalgarno Sequence (found on the ribosome) - UCCUCC
Translation in Prokaryotes • The large subunit will attach and attract the first tRNA molecule. Loading in at the P-site of the ribosome.
Repairing Mistakes in DNA Synthesis • Replication forks work at 50 bases per second • Errors = one mistake per billion • HUMAN REPLICATION • 6 billion nucleotides • Cells are replicated to create trillions of cells
DNA Polymerase Proofreading in Prokaryotes • DNA polymerase acts as an exonuclease – (an enzyme that removes nucleotides from DNA) • DNA polymerase III can remove nucleotides only from the 3’ end of the DNA, and only if they are not hydrogen bonded to a base on the complementary strand. • If a wrong base is added during DNA synthesis, the enzyme pauses, removes the mismatched base that was just added, and then proceeds with synthesis.
Eukaryotic DNA polymerases • Have the same type of proofreading ability – reduces error rate to about 1 in 10 million bases. • At this rate there would be 600 mistakes every time a human cell replicated
Three Types of DNA Repair • 1. Mismatch Repair • 2. Thymine Dimer Repair • 3. Excision Repair
Mismatch Repair • When DNA polymerase doesn’t fix the problem, other enzymes spring into action. Responsible for “mismatch repair” • The first repair enzyme is known as mutS. • “mutatorS”
Which base is right? • Hypothesis: At the conclusion of a replication process, a methyl group is added. So the proofreading enzyme will remove the nucleotide from the unmethylated strand.
XerodermaPigmentosum: A Case Study (DNA Repair Disorder) • An autosomal recessive disease in humans. • Extreme sensitivity to UV light. Skin will develop leasion after even slight exposure to sunlight. • UV Light will cause a covalent bond to form between adjacent Thymines on a DNA strand. • Creates a kink in the secondary structure of DNA. • Causes a stall in the replication fork during replication.
The Study • Cells of “normal” individuals versus cells of XP individuals. • Exposed cells to UV radiation. • Added radioactive Thymines to the cell which should be incorporated IF repair occurs. • High amount of radioactive Thymines in the normal and virtual no radioactive thymines in the XP individuals.
DNA Excision Repair • Uvr A, Uvr B, Uvr C, and Uvr D • “Ultraviolet Light Repair”
Direct DNA repair DNA photolyase
DNA Mismatch Repair and Cancer(DNA Repair Disorder) • Colon Cancer Variation = Hereditary nonpolyposis colorectal cancer (HNPCC) runs in families • Tumors will develop on the colon, ovary, and other organs by the age of 50 • 1990 – researchers mapped the susceptibility to an area on chromosome 2
Do humans have mismatch repair genes? • The research accelerated when mutS gene was identified and then research found a similar gene in a yeast genome. • The genes were so similar, they called them homologous. • Using the sequences from the genes, they located a similar sequence in the human gene – known as hMSH (human mutS homolog) • Mapped to the same region on chromosome 2 as the HNPCC susceptibility gene.
Link between cancer and mismatch repair • Cells from these patients have a mutation rate 100 times the normal. • People who inherit a nonfunctional copy of the hMSH gene have a genetic predisposition for developing HNPCC. • Evidence: Individuals who have this form of colon cancer have uneven repeats of sequences in their DNA (usually fixed in DNA repair).
Ataxia Telangiectasis (AT) • Defect in the enzyme KINASE. • Cells proceed through the checkpoints. (high mutation rate) • Radiation Sensitivity • Increased risk of breast cancer. • Any problems?
Potential Benefits to this research • If individuals with mutant forms of hMSH can be identified early in life, dietary changes and therapy could significantly reduce their risk of developing cancer
Semi-conservative Replication • Meselson and Stahl Experiment
