7.014 Handout Biochemical Genetics MIT Department of Biology7.014 Introductory Biology, Spring 2005Biochemical Genetics Introduction and Background We have learned that proteins can act as enzymes and catalyze all sorts of reactions in the cell. We discussed a biochemical approach to understanding cellular processes by purifying enzymes, determining their structure, and then studying the reactions they catalyze. Often though this type of study does not answer the question, "what does this protein do within the organism?" One approach to this question would be to remove the protein being studied and determine how this affects the organism. But, is this possible? The information required to make an enzyme is encoded as a gene in the DNA of the organism. In general, one gene corresponds to one polypeptide (a gene can also correspond to an RNA, but this is not relevant to the discussion at hand). This correspondence and advances in DNA technology allow researchers to create an organism that lacks a particular protein by removing the DNA that encodes that protein. In effect, a specific mutant organism can be created and by studying this mutant we can study the effects of removing a single protein. Even before this was a possibility, genetics played a key role in understanding many basic cellular processes. If you wanted to study olfaction but you had not identified any of the proteins involved, how could you begin? Because one gene corresponds to one protein and the genotype of an organism is often reflected in its phenotype, you could start by finding mutants that can not detect odors. The assumption would be that each mutant carries a change in the DNA that encodes a particular protein important in olfaction. The altered protein could have an altered function or be so changed that it no longer functions at all. The change in the DNA sequence is called a mutation, and the cell or organism carrying a mutation is called a mutant. Mutants with a disruption in the olfaction process would all have the same phenotype, an inability to sense odors. This is true even if each mutant had an alteration in a different gene. For example, assume that the ability to perceive odors requires three different proteins and protein 1 is encoded by gene1, protein 2 is encoded by gene 2, etc. The phenotype of an organism with a mutation in gene 1 would be the same as the phenotype of an organism with a mutation in a gene 2; neither of these mutants would be able to perceive an odor. Thus even without any knowledge of the genes or the proteins involved in a particular cellular process, we can begin to study it by collecting mutants that all display the same phenotype. A Model System For this introductory illustration of biochemical genetics we will begin with a model system. Model systems are organisms that can be easily manipulated in a laboratory environment. Widely used as model organisms are single-celled eukaryotes of the yeast family. These yeasts, like Saccharomyces cerevisiae or Schizosaccharomyces pombe are associated with the making of beer and bread. These cells are easily and quickly grown in a laboratory, can live in either a haploid or a diploid state, and can reproduce sexually or asexually. Yeast cells can be treated with chemicals or irradiated to increase the mutation rate, and they can be rather easily convinced to accept exogenous DNA. To explore more about yeast see: http://enpc1644.eas.asu.edu/modsum/YstSUM.htm. 2The yeast life cycle.Yeast cells reproduce asexually simply through mitosis where the mother cell divides to produce two identical daughter cells. If the yeast cell is diploid, it can instead enter a meiotic pathway and produce four haploid cells or "spores". Interestingly, each of these haploid spores can enter a mitotic cycle and indefinitely produce identical haploid daughter cells. Two haploid yeast cells can fuse or "mate" to create a new diploid cell. Growing yeast cells. Yeast cells can be grown easily in the laboratory. Wild-type yeast cells are prototrophs. This means that they can synthesize all the necessary cellular components de novo if provided with some basic carbohydrates and salts. The basic required carbohydrates and salts are supplied as minimal medium. Yeast cells can be grown in test tubes containing liquid minimal medium or on petri plates containing a solid form of minimal medium. A single yeast cell can only be seen with a microscope, but a single cell can divide into 108 identical cells in 48 hours. 108 cells are enough to turn a tube of liquid medium cloudy, and if the tube of medium is allowed to settle, a coating of cells is visible at the bottom of the tube. When a single cell divides on solid medium, all the daughter cells stay grouped together. 108 cells grouped together form a colony about as big as the following dot. • Yeast cells can also be grown in or on rich medium. Rich medium contains carbohydrates and salts and all the amino acids and other nutrients that yeast cells could need. When presented with amino acid supplements (or other nutrients), yeast cells do not invest cellular energy to make them de novo, but instead scavenge them from the medium. When a mutation occurs in the genome of a yeast cell that eliminates a protein required for biosynthesis, the mutant yeast cell can no longer grow on mininal medium. Such mutants are called auxotrophs. If the mutation damaged a gene encoding an enzyme needed for arginine synthesis, then the mutant cell would not grow on minimal medium but would grow on minimal media that was supplemented with arginine. Alternatively, if the mutation damaged a gene encoding an enzyme needed for nicotinic acid synthesis, then the mutant cell would not grow on minimal medium but would grow on minimal medium that was supplemented with nicotinic acid. Both the arginine auxotroph and the nicotonic acid auxotroph could grow on rich medium. Making and Identifying Mutants Mutations like those discussed above are naturally occurring due errors in mitosis or meiosis. However, to study a biochemical pathway using genetics, many mutants are needed. To this end, wild-type yeast cells can be mutangenized by irradiation or with chemicals that damage DNA. In a mutagenesis, the starting cells are usually haploid, and are treated with levels of mutagen such that most cells carry a single mutation. Why would you want to begin with a haploid strain? You are hoping to identify cells that are
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