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Lecture 18

Lecture 18-23

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Western University
Biology 1002B
Tom Haffie

Biology 1200b – Test Three Will Burke Lecture 18 – Cancer In Canada, cancer is the leading cause of non-accidental death. Men are at a higher risk than women. The top four most common are prostate, breast, lung and colon cancer. Heritability estimates from twin studies show that there is a rather low correlation (about 0.27-0.42). Embryogenesis involves rapidly diving cells. The cell cycle involves a complex called CDK (cyclin dependant kinase) which is a checkpoint that ensures damaged cells do not replicate their DNA. Cyclins are produced early in the cell cycle where they bond with CDK, which then phosphorylates targets downstream and releases the G1 checkpoint. Different sets of cyclins are used for each checkpoint. Expression of proto-oncogenes like EGF promotes cell cycling, which are sometimes treated as the cause of cancer. They are, however, genes that are required for cell division but may play a role in the rapid- cell division involved in cancer. The regulation of cell division can be caused by many different genes and proteins that are involved in the translation of signals from the EGF. Expression of tumor suppressor genes slow cell cycling. TP53 is a master tumour suppressor gene that codes a transcription factor whose activity can result in increased DNA repair, cell cycle arrest by blocking cyclin/CDK and apoptosis (cell death). Sporadic cancer requires loss of function mutations in both alleles that code for regular cell development. This is very rare as it involves both alleles to mutate. It is more common through inheritance since one damaged repressor may be from either parent. Inappropriate expression of miRNA can promote cycling, for example oncomirs. Different kinds of tumors have different kinds of miRNA expression and this is diagnostic. Cancer is deregulation. Uncontrolled growth can arise from upsetting the balance between the activities of gene products that promote cell cycling versus those products that suppress cell cycling. Cancer is also progressive. Various steps have to happen for cancer to be expressed. Cancer may begin as alterations to gene expression in stem cells. Most tissues contain stem cells, which are called pluripotent. These cells can differentiate into many different types. When a stem cell divides it creates one differentiated cell and one stem cell. Cells are not the same in tumors. Some are proliferative and some are not. A mouse family with high risk for brain tumor has one defective tumor suppressor. A nucleus is removed from a mouse embryo and replaced with a tumor nucleus and then given an electric shock. The cell then divides under the control of a tumor nucleus and creates a mouse. Thus the maternal egg reprograms the tumor nucleus. This means cancer is perhaps epigenetic. Is cancer contagious? Feline leukemia virus, mouse mammary tumor virus and HPV (human papillomavirus) are contagious. HPV leads to cervical cancer. It is a DNA virus and many different strains cause increased growth in different tissues in men and women. The number of new partners greatly increases chance of acquiring the HPV virus. Lecture 19 – Molecular Homology Molecular evolution is the study of evolution at the level of nucleic acid and amino acid. Gene evolution is the study of how genes change over time. Changes in genes that lead to evolutionary change can be mutation (insertion, deletion, frameshift), duplication, rearrangement and loss. All of these change phenotype if they’re going to have an evolutionary effect. Some mutations do not change phenotype. We will ask the question “how a mutation to a gene causes an enzyme to change substrate specificity”. Change in phenotype leads to selection. Homology has many definitions. In this course it means common ancestry. For example, the structure of a flipper and wing come from a common ancestor and are thus homologous, even though they are not completely similar. How do we know that they share a common ancestor? The gene GlsA in Volvox and Chlamy is homologous, but they do not have the same nucleotide sequence, amino acid sequence, length or function. Genome annotation involves attaching biological meaning to a sequence of DNA. This results in gene prediction, detection of regulatory elements, finding biological functions through similarity searches and can be done automatically using algorithms. Protein-coding gene prediction is used to detect what protein a gene codes for. There is a computer algorithm that can be used to detect promoter elements, intron/exon boundaries and other conserved DNA motifs. The computer sequence can splice out exons to create a deduced protein coding sequence. Protein prediction involves translating all possible reading frames of the gene and detecting which is the longest with few stops and starts. Chlamydomonas has 15,000 predicted proteins. These are not definite but are likely. More work is then done on the predicted proteins. Similar sequences are then researched. The National Center for Biotechnology Information contains a Genbank with a sequence database. About 23,500 total genomes have been fully mapped out. Sequences can be arranged to show regions of similarity and thus used to detect functional and structural similarity as well as evolutionary relatedness. There are different kinds of alignments. Global alignment involves precise similarity between two sequences. Local alignment doesn’t force two sequences to align perfectly but rather looks for different regions of high similarity. There are 155 gene sequences at GlsA that are very similar to the Chlamy gene. The Volvox gene is extremely similar, but there are others that have some similarity to certain parts of the sequence. BLAST analysis shows that there are differences in the nucleotide alignments and amino acid alignments between Chlamy and Volvox, and thus neither of these are the definition of homology. They also do not have the same length or even function. Amino acid sequence comparisons are more informative than nucleotide. 1. Nucleotides are a four letter alphabet and when converted into bits each base has two bits on information (A00, G01, C10, T11). If I is the total information in a message with G symbols written in an alphabet of n letters, I = (Gln(n))/ln2. There are 20 amino acids and thus 20 characters in the amino acid “alphabet”. 2. The genetic code is redundant. There are 64 possible triplets but only 20 amino acids as some codons code for the same amino acid. Amino acid sequence is more highly conserved. Different nucleotide sequences can be translated into the exact same amino acid sequence. 3. DNA databases are much larger. This is a bad thing because there is lots of junk, while the amino acid database is more refined. Homology determination is based on probability. There is no way to say with absolute certainty whether two genes are homologous, it is simply a conjecture. There is no way to know what the common ancestor of Volvox and Chlamy to test its genetic code. Thus decisions are based on similarity numerically and correlated with probability. The higher the similarity between two sequences the lower the probability that they originated independently of each other and became similar by chance. E-Value – The lower the E-value the greater the likelihood of real homology. The Chlamy sequence of GlsA and Volvox sequence of the same have an E-Value of 0.0, meaning the chances of these sequences developing separately is almost impossible. Lecture 21: Experimental Evolution Charles Darwin, 1859 – “In looking for the gradations by which an organ in any species has been perfected, we ought to look exclusively to its lineal ancestors; but this is scarcely ever possible, and we are forced in each case to look to species of the same group that is to the collateral descendants from the same original parent form”. Volvox and Chlamy are collateral descendants. We can try and figure out what happened in the past by how they look today which involves a lot of inference. Instead we can use experimental evolution, which is testing hypothesis about evolution using controlled experiments. Species in the lab are subjected to different conditions and observed on how they adapt over time. Model systems for EE are viruses, bacteria, Chlamy, drosophila and yeast. These species work because they reproduce very quickly (short generation time). Chlamy have about a nine hour generation time before they divide. Thus these species can be used to test evolution over a relatively short period of time. Genetic novelty can appear due to spontaneous mutation, which is just a mutation which randomly happens. Many of these are deleterious but occasionally one that is advantageous can occur. Another way is through gene duplication which occurs when a region containing a gene is duplicated. Most of the times one of these copies become destroyed and thus has no effect, but on occasion the second copy is retained. When this occurs there is more freedom for the second copy of the gene to mutate and change because it is not absolutely essential to the cell. Neo-functionalization means one copy of the gene is able to do something advantageous. Sub-functionalization means the gene is not changed but the promoter region is, changing the conditions in which the gene is expressed. Gene rearrangement involves genetic processes that rearrange the genome. A promoter which is gene dependant can move to promote a different gene than it originally did, which creates genetic novelty. The Long Term Evolution Experiment asked “Can evolution produce adaptation if it depends on random mutations, most of which are harmful?” They used E. coli to reduce complexity (asexual reproduction, no recombination). Any change over time is due to duplication, spontaneous or rearrangement. The population size for the experiment was huge. The experiment started with 12 identical populations all coming from one original bacterium and are thus genetically identical. Every day .1mL of the culture is put into fresh media so the cells can continue to grow. This is done every day for all the populations. Every 500 generations (75 days) a sample can be removed, frozen and later compared to another generation. After about 30,000 generations one of the populations is more turbid (more cells for mL) than the others. The cells in that culture had evolved the ability to use citrate as a carbon source. The genome of E. coli is about 4.6 million bp, and pretty much every mutation was tried many times. The one that allowed the culture to utilize citrate was very rare. Citrate was only in the solution to keep iron available for the cells. The cells normally have no ability to bring in citrate under normal conditions. The glucose available runs out after about 8 hours and the remainder of their time is spent in stationary phase. It represents an ecological opportunity for the species to use citrate instead for growth. Since all of the generations still were kept frozen the ancestors can be thawed out and used to find when the mutation occurred. One question is whether the mutation was contingent on another mutation that occurred prior. This was tested by taking prior generations and having them grow on a citrate agar. It was found that some generations prior to 30,000 were able to grow. This shows that there was more than one mutation – one that allowed the cells to utilize the citrate, and another that allowed them to utilize it at extremely fast rates. The evolution in the line can be replayed. Cells can be taken from different generations and grown for another many generations to see if the same mutations occur. When this was tested it was found that before 20,000 generations there was no way for this capability to arise. This is proof that another mutation occurred prior to 20,000 that the later mutations were contingent upon. The results of this experiment showed that there are three stages of a mutation – potentiation (an unknown mutation that the final result was contingent on), actualization (the adaptation is observable), refinement (species is able to fully utilize change). The actual mutation that occurred in the E. coli experiment occurred on the citT gene. In the actualization step of the experiment gene duplication duplicated citT and placed it downstream of the rnk promoter which is a very strong promoter that is always on, especially in the presence of oxygen. This means the citT gene is expressed. + The refinement of the Cit mutation occurs when there is an increase in the number of rnk-citT modules. This creates a very strong Cit phenotype due to duplication. The more modules there are the greater the culture density. Mutations in each of the reply experiments showed that the amplification length was slightly different + but still came up with same Cit phenotype. The new line of E. coli can grow on citrate. The defining characteristic of E. coli is that it cannot grow on - citrate. Does this new line warrant being considered a new species? Cit was not driven to extinction and it was found that they are more efficient at utilizing glucose, meaning they have their own ecological niche. Lecture 20 - Molecular Convergence There can be differences in the amino acid sequence between two homologous genes due to the huge amount of time since the species diverged. These single amino acid changes are normally neutral and do not change the overall phenotype. The neutral theory of molecular evolution notes that lots of mutations have no effect on the protein at all. They are random and are inherited but are neither deleterious nor advantageous. The number of differences in protein sequence between different species is roughly proportional to the time since the species diverged. To be more specific, as the number of millions of years since divergence increases, the number of amino acid substitutions per 100 residues increases linearly. Some genes diverge more quickly than others. There is a relatively constant rate of change for gene differentiation. The neutral theory explains the linear rate of change for genes, which is opposed to natural selection, by calling most mutations neutral and thus not selected for or against. This linear graph can be used to calculate the millions of years since divergence by testing the number of amino acid substitutions per 100 residues. This creates a molecular clock. Testing the numb
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