Friedrich Miescher – Isolateds nuclei from WBC in pus. • Acidic substance found – containing nitrogen and phosphorus. • Name given = nuclein
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.
Smooth bacteria have a polysaccharide coat that appears to be necessary for infection. Heating will destroy the coat, but not the DNA.
Transformation is the intake of external molecules from an outside source. It is now associated with “naked” DNA. Conclusion: DNA is the transforming factor! NOT PROTEINS!
Beadle and Tatum One Gene – One Enzyme hypothesis : the Function of one gene is to dictate the production of a specific enzyme. The final enzyme is responsible for growth…
Deciphering the Structure of DNA • Phoebus Levene – • 1909 – Ribose is present in some nucleic acids • 1929 – Deoxyribose is discovered in others
Mutation Vocabulary Gene Mutation Chromosomal Mutation Duplication Deletion Translocation Inversion • Point Mutation • Substitution • Silent • Missense • Nonsense • Frameshift • Insertion • Deletion
Components of Transcription • Initiation • Promoter • Sigma Factor • RNA Polymerase • DNA Template Strand • Elongation • RNA Nucleotides • Termination • Rho Protein • Terminator Sequence
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. (TTGACA…..TATAAT) Memorize this sequence!!
Promoter sequence is found on the Coding Strand • The mRNA sequence is complementary to the Template strand, though • The sigma appeared to be responsible for guiding the RNA polymerase to specific locations. • The sigma will release once initiation has commenced. • Roughly 15 nucleotides into the transcription process
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 TATA box.
Termination of transcription process • Rho Independent • Hairpin Loop • Inverted repeats creates a pairing on the single strand • AGCCCGCC ………….GGCGGGCT • Followed by a long series of UUUUUUUUUU -- Causes the RNA polymerase to cleave the transcript • Rho Dependent • RNA polymerase stalls over the termination sequence • Rho enzyme catches up to RNA polymerase..bindsto the enzyme, which causes cleavage to occur.
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
Prokaryote vs. Eukaryote • Prokaryotes have the Shine Dalgarno sequence because prokaryotes will place multiple genes on ONE mRNA strand along with multiple AUG sequences embedded within the gene. • The shine-dalgarno sequence will establish which AUG sequence is the true “initiator AUG” • Eukaryotes only make 1 gene mRNA sequence, so no Shine Dalgarno sequence is used.
Translation in Prokaryotes • The large subunit will attach and attract the first tRNA molecule. Loading in at the P-site of the ribosome.
PCR – Directing DNA Replication • 1. Figure out the DNA sequence • 2. Two Types of DNA Primers (Forward and Reverse Primers) • 3. A large supply of DNA nucleotides • 4. Taq1 polymerase – a specific type of polymerase found in hot spring microbes
The Process • 1. Heat • 2. Primers and DNA polymerase are added (cooled) • 3. Single nucleotides then are mixed • 4. Cycle continues
How much can be made? • 2n • n = the number of temperature cycles
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 lesions 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
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)
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. (hereditary colon cancer) • 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?