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Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
Terms in classical genetics
I B. DEFINITIONS AND DISCUSSION OF TRADITIONAL GENETICS
Since the crux of any field is in an understanding of both the denotations and connotations of the terms used, it is appropriate to start off with a number of definitions (and some controversy): traditionally, a mutant is a strain that has an altered growth property (termed the phenotype) relative to An arbitrarily chosen benchmark strain (termed the wild type). In this description, a mutation is the change in the DNA sequence (the genotype) which causes that altered phenotype. I would like to propose instead the following definition: a mutation is a change in the sequence of DNA from what is found in the wild type irrespective of the resulting phenotype. A strain carrying such a change is termed a mutant.
The two sets of definitions may seem identical at first blush but they are different in an important way. In the first version, the definitions hinge on an altered behavior (phenotype). This makes sense since, until very recently, that is all that could be examined easily. The second set of definitions suggests that the genotype is the crux of the matter whether or not an altered phenotype is obvious. These latter definitions are becoming increasingly functional because we are increasingly able to examine the genotype directly, through sequence analysis.
While in part deceptive (you are not going to be able to find a mutant strain unless it looks different in some way), the latter definitions stress two points:
When you analyze the phenotype of a particular mutant you learn the phenotype caused by the mutation in that strain. However, when you analyze the phenotypes of a number of mutants affected in the same gene, you begin to learn the range of functions that gene product is involved in. More importantly, the analysis tells you which mutants are typical (perhaps representative of a complete loss of gene product function, for example) and which are atypical (which may mean "interesting" or may mean "weird") for subsequent biochemical analysis. This last point brings another prejudice that will be noted throughout the text, namely, that the best use of genetics is in conjunction with biochemical analyses of the various mutant strains.
A change in genotype refers to any known alterations in the DNA sequence from that which is arbitrarily referred to as wild type ("arbitrary" as it is whatever happened to be in the strain originally isolated from nature). This genotype does not necessarily explain the growth behavior of the organism but rather is an indication of what gene or genes might be altered in it relative to wild type. For our purposes, we will define a gene as a region of DNA that encodes a product (either RNA or protein).
You should also become aware of the proper usage of genetic terminology and this will be sprinkled throughout the text. If a strain is altered in a gene whose product is itself involved in, for example, histidine biosynthesis, then the first mutation isolated in such set of genes would be called his-1 (note italicization, though underlining will often be used instead). If that mutation subsequently is determined to be in a gene called hisA, then the mutation would now be called hisA1. Different "versions" of a gene are called alleles of one another and the "1" in hisA1 is the allele number, which is used to name that particular mutant version of the his region. There should not be another mutation termed hisB1 for that organism, since that his allele number has already been used. The phenotype of an organism is noted by a different nomenclature. For example, a strain requiring histidine for optimal growth (presumably like the strain containing the hisA1 mutation) would be termed His-, while one not requiring histidine would be noted His+. The take-home lesson is that the genotype is noted by three small letters followed by either a capital letter if a gene has been designated or a dash if it is not, followed again by a mutation or allele number. All of these symbols are italicized. A phenotype, or altered growth behavior of the cell, is designated by a capital letter followed by two small letters with either a plus or minus superscript and such a designation is not italicized. When writing the genotype of a strain, only mutant loci are named (all others are assumed to be wild type). This is in contrast with the case of plasmids, where only replication functions can be assumed and any other encoded genes should be listed. Typically plasmids are assumed to have only those genes that are noted.
Mutants can either be tight or leaky: A tight mutant displays its non-wild-type phenotype distinctly and clearly while a leaky mutant displays a much less distinct phenotype compared to wild type. Mutants can also be stable or unstable and this is an indication of the frequency with which they revert to an apparently wild-type phenotype (reversion is the return of a mutant to a wild-type phenotype). We will come back to these topics again and again throughout the course.
A conditional mutant is one that it is known to display its mutant phenotype only under certain conditions. A particular condition where the mutant phenotype is evident is termed non-permissive, while the wild-type phenotype is observed when conditions are permissive. The typical examples are either cold-sensitive or temperature-sensitive mutants. These are mutants that display their mutant phenotype (in a recognizable fashion) only at low or high temperatures, respectively.
A strain that can derive all carbon requirements from the principal carbon source is termed a prototroph. If one or more other organic growth factors (like amino acids, nucleotides, or vitamins) are required by the strain, it is termed an auxotroph. Occasionally reference will be made to conditional lethal mutations where the strain carrying such a mutation dies under the non-permissive conditions regardless of the medium. Such mutations are typically found in genes whose products perform DNA replication, RNA transcription, protein synthesis or other functions essential on any media.
A final point on the arbitrariness of phenotypes: when a strain is referred to as His-, you may assume it requires the addition of histidine to the media for good growth. You should not assume that it fails to make any of its own histidine, simply that it does not make enough for optimal growth under the conditions tested. Similarly, a strain might make rather less histidine than a wild-type strain, but if it is capable of making sufficient histidine so that it grows normally without supplementation, it is designated His+. Again, the assignment of either a His+ or His- phenotype is purely arbitrary according to the actual conditions of growth used in the experiment.
AN IMPORTANT SUGGESTION
To the extent that the experience of previous students is instructive, it appears to be important that you develop mental pictures of the biochemical processes that we are analyzing genetically. For example, the frequency of occurrence of temperature-sensitive mutations, relative to that of a "loss-of-function" mutations, will seem arbitrary until you picture a protein and imagine the sort of alterations necessary to make that gene product "fail" at elevated temperature while it continues to function at lower temperatures. Such an effect on the protein clearly implies an alteration of the gene product, not its destruction. It should also seem reasonable that there are not too many different alterations (mutations resulting in amino acid changes) that will have this property and therefore mutants with such phenotypes should be rather rare compared to alterations that destroy activity at all temperatures since there will be many more alterations that will have that result. In a similar vein, it should seem reasonable that restoration of normal function to the affected gene products should result from similarly rare, very particular changes in the amino acid sequence (resulting from changes in the genotype). Without such mental pictures of the biochemistry underlying the phenotypes, genetic analysis will seem to be an arbitrary and formal game. With such images, the genetic result will "make sense" because it reflects the biochemical reality.
When thinking about the problems, it might be useful to consider three "levels": (i) The genotype, which directly affects the production and function of gene products; the functionality of these gene products can be viewed as a (ii) "biochemical phenotype" which then determines the (iii) growth phenotype of the organism. Considering the middle level should make it easier to see the connection between the genotype and the growth behavior of the bacterium.
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