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Genetic Methods Introduction

The genetic systems found in bacteria and fungi are particularly powerful. The small size of the genome (all the genetic material in an organism), the ability to examine both haploid and diploid forms, and the ease of large-scale screens have made them the method of choice for many investigations. Some of the key features will be summarized in this section.

Microorganisms such as bacteria and fungi have several advantages for genetic analysis. They have a haploid genome, thus an investigator can detect recessive phenotypes easily and rapidly. In the haploid (1N) state, only one allele is present for each gene, and thus its phenotype is the one observed in the organism.

Bacteria can carry plasmids and can be infected with viruses, each of which are capable of carrying copies of bacterial genes. Thus bacteria can be partially diploid, or merodiploid, for some genes. This allows one to test whether alleles are dominant or recessive. 

Bacteria are capable of sexual transfer of genetic information, during which time homologous chromosomes can recombine. Thus one can use recombination frequency to map genes, analogous to the process in diploid sexual organisms. Indeed, a high frequency of recombination was essential in investigations of the fine structure of genes. Bacteria grow, or increase in cell number, very rapidly. Generation times can be as short as 20 to 30 minutes. Thus many generations can be examined in a short time.

An investigator can obtain large quantities of mutant organisms for biochemical fractionation.

Bacterial genomes are small, ranging from about 0.580 (Mycoplasma genitalium) to 4.639 million base pairs (E. coli), with about 500 to 4300 genes, respectively. Compared to organisms with genomes 100 to 1000 times larger, this makes it easier to saturate the genome with mutations that disrupt some physiological process. Also, the smaller genome size, plus the availability of transducing phage, made it possible to isolate bacterial genes for intensive study.

Genomes of several bacteria are now completely sequenced, so all the genes, and their DNA sequences are known.

Yeast, such as Saccharomyces cerevisiae, are eukaryotic microorganisms that have both a haploid and a diploid phase to their life cycle, and thus have these same advantages as bacteria. Although its genome is larger (12 million base pairs), and it has 16 chromosomes, it is a powerful model organism for genetic and biochemical investigation of many aspects of molecular and cell biology. The genome of Saccharomyces cerevisiaeis completely sequenced, revealing about 6100 genes.

One can use mutagens to increase the number of mutations, e.g. to modify bases, intercalate, etc. Specific mutagens will be considered in Part Two of the course.

Replica plating allows one to test colonies under different growth conditions. This is illustrated in Fig. 1.8 for finding mutant with new growth factor requirements. Replica plating can be used to compare growth of cells on complete medium, minimal medium, and minimal medium supplemented with a specific growth factor, e.g. an amino acid like Arg (the abbreviation for arginine). Cells that grow on minimal medium supplemented with Arg, but not on minimal medium are Arg auxotrophs. The word auxotrophmeans "increased growth requirements". These are cells that require some additional nutrient (growth factor) to grow. Prototrophs(usually the wild type cells) do not have the need for the additional factor and grow on minimal medium. In this case, they still make their own Arg.

 

           

Figure 1.8. Replica plating of microorganisms.  Panel A shows the technique of replica plating to screen for drug sensitivity. Panel B illustrates its application to finding mutants with growth factor requirements.

Sometimes the trait one is selecting for is lethal to the organism. In this situation, one can screen for conditional mutants. These are mutants that grow under one condition and not under another condition.  Conditional mutants that grow at a low temperature but not at a high temperature are are called "temperature sensitive" or ts mutants. Conditional mutants are not necessarily associated with lethality. The dark ear tips, nose and feet of a Siamese cat are the phenotype of a temperature sensitive mutation in the clocus (determining fur color). The enzyme encoded is not functional at higher temperatures, but is functional at lower temperatures, such as the extremities of the cat. Hence the fur on these parts of the Siamese cat’s body is pigmented.

         

Figure 1.9. Coat color in Siamese cats is determined by a temperature sensitive mutation in an enzyme needed for pigment formation. Siamese are homozygous chch, which encodes an enzyme that is active at low temperature (in the extremities of the cat) but inactive elsewhere.