Test 2: Discussion Flashcards
Triplet Binding assay of Nirenberg & nature of the Code:
Nirenberg’s research allowed for the sequencing of the genetic code. He used ribosomes, charged tRNA (carrying amino acids), and template RNA (mRNA). One amino acid was radiolabeled per reaction and he used a filter that would allow for unbound tRNAs to pass through. However, ribosomes and ribosomes bound to radiolabeled tRNA would not pass through the membrane. Since codon compositions were known (not exact sequences) it was possible to narrow the decision as to which amino acids should be tested for each specific triplet. Nirenberg added specific trinucleotide RNA plus all of the possible charged tRNAs (one radio labeled/reaction) and the ribosome with the bound triplet that was caught in the filter were analyzed for radioactivity. If radioactivity was detected it meant that the charged tRNA was bound to the RNA triplet associated with the ribosome. Results showed that there were 64 different codon possibilities for triplet codes, 61 of the possible codons specifiy amino acids, 3 codons signal stop, one codon specified Met & start, the code was unambiguous (each codon only ONE meaning), and the code was degenerate (more than one way to specify an amino acid).
Types of Info that lead to hypothesis of Protein being genetic material:
Proteins were thought to be genetic material b/c proteins have 20 different amino acids as their “building blocks”. Where DNA only has 4 nucleotides as its building blocks. DNA was thought to be to “simple” to be able to carry out the functions of the unknown genetic material at the time. Also protein’s stability are highly variable as opposed to DNA which are very stable.
2 experiments that disproved that hypothesis:
Avert et al.:
Read Griffith’s transforming principal using strains of pneumonia and realized that the identification of this principle would tell them what genetic material was. IIR was the avirulent strain and IIIS was the virulent strain used. A crude extract of IIIS was combined with IIR and that resulted in both IIR & IIIS being present. They then used 3 plates with IIIS and added protease, RNAses, & DNAses to one plate each. IIR was then added to each separate enzyme plate and waited to allow for replication to occur. In the proteinase & RNAse plate, both strains were growing which meant that transformation had occurred. In the DNAse plate only IIR was growing which meant that transformation had NOT occurred. This proved that DNA was the genetic material b/c it showed that when the enzyme destructed the DNA, transformation didn’t occur.
Hershey & Chase:
They used the knowledge that viruses were made up of nothing but genetic material and a coat. They knew that the genetic material was made up of proteins & DNA but did not know which was inserted into an object when an infection occurred. They also knew that whatever was inserted were the genetics and that was how a coat was formed on the new infected object. Hershey & Chase used the known facts that proteins were composed high conc. of Sulfur & that DNA was composed of high conc. of Phosphorus to determine what was being inserted by the virus. Radio labeled [P] & [S] were added to 2 separate cultures containing a virus. The virus was allowed to replicate and then both samples were transferred to separate hosts with no label. After viewing with an electron microscope, they found that the radio labeled protein [S] was outside of the cell and the radio labeled DNA [P] was inside the cell. Because of their findings, DNA was proven to be the genetic material.
One Gene One-Enzyme Hypothesis:
Beadle & Tatum worked with Neurospora b/c it grows on minimal medium and it has abundance of metabolic pathways. They induced a nutritional mutation on the Neurospora during the haploid phase to form auxotrophs. This proved that each different mutant was due to the block of pathway at different points.
Levels of Protein Structure:
Primary: linear arrangement of amino acids
- directly determined by gene sequence
- most impt. b/c it impacts all subsequent levels
Secondary: hydrogen bonding between peptide bond components
- alpha helix: 3D helix
- beta pleated sheet: 2D
Tertiary: 3D arrangement of chains
- determined by R-group interactions
- direct affect on functionality
- affected by disulfide bridges, ionic bonds, hydrogen bonds, & hydrophobic interactions
Fundamental basis of high fidelity copying:
The fundamental basis for high fidelity copying is the complementarity of the nucleotides due to the hydrogen bones. Adenine & Thymine consist of 2 hydrogen bonds while Guanine & Cytosine consist of 3 hydrogen bonds and as a result G-C is more stable than A-T.
Details of Replication in Prokaryotes:
Replication begins when proteins called helicases denature the double helix by breaking the hydrogen bonds & dissociating the antiparallel strands. As this happens, single strand binding proteins stabilize the open strand and prevent stand association. Due to the unwinding of the strands a coiling tension (super coiling) is created ahead of the replication fork. Super coiling can be relieved by gyrases (enzyme that is a member of topoisomerases). Primase then synthesizes the RNA primer which provides the free 3’ OH for DNA polymerase. DNA polymerase then synthesizes the nucleotides in the 5’->3’ direction. As the 2 strands unwind and the replication fork continues down the helix, synthesis is continuous on the leading strand going 5’->3’ from the origin of replication towards the replication fork while synthesis is discontinuous on the lagging strand which is going 5’->3’ away from the replication fork. The lagging strand produces small pieces of fragments called Okazaki fragments. The RNA primer is then replaced with DNA polymerase I followed by the Okazaki fragments being joined together by DNA ligase to complete the final closure.
Watson & Crick model of DNA, including composition, structural aspects and features that allow it to serve as the genetic material:
Watson & Crick were the first to accommodate all of the DNA data that had been discovered before them. They proposed that DNA was a right-handed double stranded alpha helical structure of antiparallel polynucleotide chains that are composed of two types of complimentary nucleotides. Phosphodiester bonds link the adjacent nucleotides and the strand directionality consisted of a 5’ phosphate at one end and a 3’ hydroxyl (OH) at the opposite end. More specifically, that the 2 types of nucleotides were purines and pyrimidines. Purines (Adenine/Guanine) have double ringed nitrogenous base, one is 6 membered rings while the other is 5 membered, and both rings have nitrogen substitutions in them. Pyrimidines (Thymine/Cytosine) are single six membered ring with nitrogen substitution in the ring. They also proposed that the complementarity of the bases were due to the 2 hydrogen bonds between A-T and the 3 hydrogen bonds between G-C. The component parts of all 4 of the nucleotides were a phosphate group, a 5-carbon sugar, and a nitrogenous base. This helped explain the high molecular stability of DNA. The hydrogen bonds are weak but millions to billions of them provide for an overall strong bond. The sugar/phosphate portions provided a hydrophilic outer wall and the hydrophobic nitrogen bases are sheltered in the core. It was also discovered that each turn of the helix occurred every 10 base pairs. The model Watson & Crick proposed was known as B-DNA, which is the most common form. However, variations in DNA structures were later discovered.
Compare & Contrast autopolyploidy and allopolyploidy, focusing on causation, impact on the organism, and the long-term consequences in nature:
Both are a form of euploidy that occur more commonly in plants and both forms require a cold shock during cell division which disrupts the spindles resulting in the cells to revert back to interphase where an S-phase DNA replication occurs again. Autopolyploidy pertains to a single species and usually results in larger leaves, fruits, flowerers, etc.… However, this also leads to the plant being sterile which results in a dead end in nature. Allopolyploidy involves the hybridization of 2 different species prior to the cold shock. These offspring are not sterile but are typically reproductively isolated. If allopolyploidy produces another mate for the offspring, instantaneous speciation can give rise. The chances of allopolyploidy are also much less likely to occur compared to autopolyploidy due to the unlikely events that must occur just right.
