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1200b Test Two.docx

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Biology 1002B
Tom Haffie

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Biology 1200b – Test Two Will Burke Lecture 10 – Evolution of Eukaryotes All morphologically complex life is eukaryotic. All eukaryotes share common complex traits – nucleus, trafficking, cytoskeleton, sex, phagocytosis, organelles. Prokaryotes show almost no tendency to develop this kind of complexity. If evolution through natural selection is gradual then why don’t these simple organisms evolve? The evolution of eukaryotes was driven by oxygen. The earliest bacteria were anaerobic (four billion years ago) and as such used fermentation to continue glycolysis. This produces very little ATP while still breaking down glucose. About 2 billion years ago cyanobacteria evolved. These bacteria can split H O into oxygen in a process 2 called oxygenic photosynthesis. They are the only single celled organisms that can evolve CO and2are responsible for oxygen in the atmosphere. This led to aerobic respiration. Oxidative phosphorylation in bacteria occurs on the cellular membrane. The problem with this is that the larger a bacteria becomes (more centers of phosphorylation) the more proteins and energy is required for support. Volume increases much faster than surface area and soon the cell will not be able to support its own volume. Eukaryotic cells have very small plasma membrane surface area to volume ratio. This is not a problem since these cells have mitochondria internalized. Eukaryotes as such have far more energy and therefore code for many more proteins than prokaryotes. Genome size varies by huge amounts amongst eukaryotes but is far greater than prokaryotes (eukaryotes have the ATP to support it). Primitive cells developed an endoplasmic membrane (reticulum) from the infolding of the plasma membrane. Cells developed a nuclear envelope to separate DNA from the rest of the cell in order to control transcription. Cells then took in mitochondria (and later chloroplasts) through endosymbiosis. Mitochondria are thought to have formerly been an aerobic bacterium. Chloroplast would have been cyanobacteria. Evidence for endosymbiosis – Morphology (mitochondria and chloroplast look like cyanobacteria), formation (division of these organelles is very similar to bacteria), electron transport chains (only chloroplast and mitochondria have these components), genomes (mitochondria and chloroplast have their own genomes and can synthesise proteins). The mitochondria must become fully part of the developing cell. One belief is that over millions of years genes have moved from the organelles to the nucleus in a process called lateral gene transfer. The function of the transferred gene is still retained. Southern blotting is used to test if a genome has a particular gene and how many copies. Genomic DNA is isolated and ran on a gel. A DNA probe hybridizes with the single stranded DNA to indicate that this sequence is similar to the probe. Thus a certain gene can be detected. DNA can be isolated from the mitochondria and run on a gel. A protein that is mitochondrial can be tested as being coded from either the mitochondrial or nuclear genome. Some eukaryotes do not have mitochondria. These tend to cause disease and are dangerous. These are not evolutionary intermediates between bacteria and eukaryotes. cpn60 is a mitochondrial protein that is absolutely essential and is still found in giardia, a mitochondrial-less eukaryote. This shows that giardia evolved from mitochondrial eukaryotes and that lateral gene transfer of cpn60 occurred very early in evolution. Lecture 11 – Intro to Prokaryotic Genes Modern endosymbiotic genomes are greatly diminished. In Chlamy there are 120,000 base pairs in the nucleus, 200 in the chloroplast and 16 in the mitochondria. Both the chloroplast and mitochondria have circular DNA. E.coli, a prokaryote, has far more kilobases (5000) than either chloroplast or mitochondria. Not all genes code for proteins. Some code for RNA (tRNA, ribosomal). Mitochondria and chloroplast have so few protein coding genes because they have no need for movement or enzymes like helicase that now occur in the cytoplasm of the cell. Redundant genes are removed by mutation and deletion or lateral gene transfer to decrease genome size as much as possible. Lateral gene transfer occurs due to coordinate control (allows the nucleus more control over the cell) and because DNA is favourably removed from organelles where ROS (reactive oxygen species) are created and liable to harm the genetic material. Nuclear DNA is different than mitochondrial in that it undergoes sexual recombination. This allows it to have a greater level of diversity and is thus more favourable. Why haven’t ALL organelle genes moved to the nucleus? Various reasons – It is too difficult to transfer from mitochondria to the nucleus, some local control might be necessary for function, some genes are too large and changes might be required for a gene to exist in a eukaryotic environment rather than a prokaryotic. Elysia are sea slugs that feed on algae called Vaucheria. They have no cell walls and are coenocytic, meaning a sea slug can simply bite off the end and absorb the entire cytoplasm of the algae. The chloroplasts from the algae enter the digestive lining of the sea slug and undergo photosynthesis meaning elysia is an autotrophic animal. mRNA (and tRNA) pairs with itself (complementary base pairing) to form structure by folding and bending. Ribosomal RNA base pairs with itself and is catalytic and structural. DNA polymerase is interesting because it replicates the genes that code for itself. In a gene the promoter is a sequence that signals for RNA polymerase when to start transcription. Lecture 12 – Prokaryotic Gene Function The start point for transcription begins downstream (-10 sequence) of a bubble that forms at the promoter sequence. The terminator sequence is transcribed into RNA and makes a hairpin structure by pairing with itself which signals polymerase to stop. Terminators have different efficiencies as they can be different lengths or be packed with G and C bases which increase bonding. Start codons are universal (same in eukaryotes as prokaryotes). The stretch of DNA between promoter and start codon is called UTR (untranslated RNA) and is transcribed but not translated. Ribosomes are made up of a small and large sub-unit. Translation initiation is stabilized by mRNA and rRNA base pairing. In bacteria there is ribosomal RNA that pairs with mRNA in a sequence called the ”SD Box”. This is a region in the DNA that base pairs with ribosomal RNA to initiate translation. It occurs upstream from the start codon within the UTR. Eventually translation stops. A protein release factor terminates translation by binding to the mRNA when tRNA does not. The genomic code is universal. This is essential for lateral gene transfer. There are only a few minor unique changes. Some base pairs code for the same codon. In prokaryotic cells mRNA is never free floating around the cell. They become translated before they are even finished being transcribed. This can create a strand of DNA with chains of mRNA all along it, while ribosomes attach to these strands and create polypeptide chains. Lecture 13 – Prokaryotic Gene Regulation What happens if gene signals (promoters, stop codons) are changed by mutation? Mutations at the promoter can either be harmful or beneficial depending on whether they increase or decrease efficiency. Mutation in the SD Box is only harmful if it is turned into a start codon. Mutating the start codon destroys it and probably kills the gene. Mutations in coding genes themselves can be silent where one codon is substituted for another and sometimes codes for the same amino acid. Alternatively the substitution may be missense where a different amino acid is created instead and can have major changes to the genes expression. Another mutation is nonsense where a codon is mutated into a stop codon which probably destroys the genes function. Indel mutations cause a shift to the frame of the gene (recognized in threes) and are very severe. Stop codons are understood by translation. Thus a mutation to the stop codon will cause the ribosome to keep translating until another stop codon. This has caused some genes to evolve redundant start codons to protect against such mutations. Trp are not translated and thus have a promoter, a terminator and an anticodon. After transcription they can bind to an mRNA with the complementary sequence. Trp is thus an operon. Operons bring several genes under the control of one promoter. Lac operon is an inducible operon. Its default state is off but in the presence of lactose, a substrate, the operon is switched on and lac y and z are created to break down the lactose. When no lac is present the lac repressor attaches to DNA and blocks transcription by creating a loop. As soon as lac binds to the repressor the gene can be expressed. Lecture 14 – Eukaryotic Gene Expression The nuclear envelope in eukaryotes is important as it keeps the ribosomes separate from mRNA until they are ready to be translated which allows various mechanisms to occur. These include transcriptional regulation (determines which genes are transferred), posttranscriptional regulation (determines types and availability of mRNA to ribosomes), translational regulation (determines rate at which proteins are made) and posttranslational regulation (determines availability of finished proteins). Nuclear gene structure is more complicated than in organelles. In eukaryotes there are proximal regions upstream from the promoter which are protein binding sites. Far upstream from the gene are enhancers which are regulatory. The nucleus has more polymerase such as RNA polymerase II which recognizes protein coding gene promoters. The polymerase only is attracted to these promoters if there is already a TATA box with a protein attached to it. A helix-turn-helix is a structural motif that binds DNA, for example the lac operon. Zinc finger DNA binding motifs are able to associate with zinc cofactors and form a specific shape. Leucine zippers hold together two DNA binding proteins. Enhancers can affect DNA from far away because the DNA bends using a co-activator that links it together activators on the promoter. This creates a promoter proximal region. This causes the promoter to be far more attractive to binding cofactors. There is no specific direction that the enhancer has to run. Unique combinations of activators control specific genes. This occurs with tissue specific activator proteins. Different activators in the eye control different genes than in the liver. One way is by changing the attractiveness of a promoter by binding proteins. Once a message is transcribed there is a phosphate cap placed on the end. The other end contains a polyadenylation signal that stops transcription in eukaryotes. Transcription continues through the polyadenylation signal which is recognized in mRNA and is cut by RNase, creating a cleavage site. This terminates transcription. A poly-A tail is added on by polymerase. At this point snRNPs (complexes of protein and RNA) are attracted to introns. snRNAs pair with themselves and associate with the intron to make a spliceosome. They form loops and are cut away and degraded. Alternative splicing occurs when a portion of RNA is either expressed as an intron or exon. For example, skeletal muscle expresses an intron that smooth muscle removes. Since this removal process does not occur in bacteria there is never a difference between protein expressions. The nuclear envelope has pores that allow different species to travel in and out. mRNA leaves through a pore into the cytoplasm where it attracts a ribosome for translation. There is no SD Box in eukaryotes. The ribosome simply scans through the RNA until it comes across a start codon and begins translation. The process is basically the same from this poi
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