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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
I E. RECOMBINATION
Throughout the sections of this text dealing with mapping, reference will be made to types of recombination. While the molecular mechanism of recombination is exceedingly complex, for our purposes a recombinational event can be thought of as double-strand breaks in each of two DNA helices followed by the reciprocal exchange and rejoining of the ends. Typically such events occur between two regions with nearly identical nucleotide sequences. Such recombination is termed homologous, legitimate or RecA-dependent recombination (since it requires, among other proteins, a functional recA gene product). This recA system works best on regions of homology more than 200 bp in length, though shorter sections of homology can be utilized at reduced frequency. Occasionally recombination events occur between regions with little or no homology. Such events do not require recA product and are called RecA-independent, non-homologous, or illegitimate. A hybrid class of recombination, which involves events only at very particular DNA sequences, is called site-specific recombination. These topics will be treated again in section IV E.
I F. THE GENETIC CODE AND ITS TRANSLATION
In considering the nature of mutations and their effect on the amino acid sequence of the product, it is appropriate to mention the genetic code and make some comments about its structure. In general, the code is set up to take advantage of what are called the "wobble" rules. That is, U has the capability, in either the 5' position of the anticodon of the tRNA or in the 3' position of the codon of the mRNA, to form hydrogen bonds with either A or G. This results in a given tRNA being able to translate two different codons so, if the code is to have specificity, those two codons must encode the same amino acid. The net effect is a code where the 3' position of the codon treats U the same as C and A the same as G with two exceptions: UGA (termination)/UGG (tryptophan) and AUA (isoleucine)/AUG (methionine). The problem is avoided in the cell in the first case by not having a tRNA to recognize termination signals and in the second case by modifying the anticodon at the U position of the tRNA so that it does not wobble and read the methionine codon.
Since the third position of the codon tends to be redundant, one typically finds that the choice of base at that position reflects the GC content of the cell. If for example an organism is GC-rich then it will tend to use G instead of A in the third position whenever the choice is possible. Similarly it will tend to use C instead of U in that position. Codon usage is non-random in other ways: even if an organism is GC-rich it is often found that the organism will choose one particular codon most of the time for encoding a particular amino acid. The reason for these non- random distributions are not obvious, but it is important for the organism to balance its codon usage with the particular levels of the different tRNA's available in the cell, not all of which are present at similar levels. It is interesting to note that "translation" (that is, the actual changing from the nucleic acid code to the amino acid code) does not take place on the ribosome but actually takes place when the aminoacyl tRNA synthetase "charges" the appropriate transfer RNA's with amino acids.
As a final complication, the reading context plays a role in the rate and therefore the efficiency of translation. Reading context refers to the sequence immediately adjacent to the translated codon, particularly the base on the 3' side. It seems that certain contexts are optimal in allowing the most rapid translation possible. Proteins required at high level tend to be encoded by optimum contexts while proteins produced at low level tend to have suboptimal contexts. Optimization of translation thus involves the use of codons with high populations of decoding tRNAs as well as the appropriate choice of adjacent codons. The practical use of this knowledge comes in the in vitro synthesis of genes for eventual high level expression of desired products.
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