Studies the behavior, biochemical structure and functions of genes in order to be able to directly intervene (manipulate) the genome of living things. From this opportunity comes the name “gene manipulation” given to this section of genetics. A new science called genetic engineering has even arisen to denote the set of technologies involving the manipulation of genetic material within and across species. This discipline has developed since 1970 when G. Khorana, J. Shapiro and others succeeded, with appropriate manipulation techniques, first to isolate a gene in its pure state, then to link it back to a precise biological function, and finally to “synthesize” it artificially, which is equivalent, within certain limits, to “creating” the elementary basis of life.
The development of these techniques, which are based on knowledge of molecular biology, is so potentially disruptive that it led J. Shapiro to withdraw from such scientific work soon after his data were published. Thanks to new methodologies related to genetic manipulation and the ability to rapidly sequence DNA, genetics has undergone tremendous development in both basic knowledge and possible practical applications. With mutation analysis of many genes, it has been possible to study their function in depth and also to identify the genes responsible for many genetically transmissible diseases.
Genes from complex organisms can be inserted into simpler organisms, and their role and structural features can be analyzed in great detail. The brewer’s yeast Saccharomyces cerevisiae represents one of the most studied genetic systems in recent years. It is a single-cell eukaryotic organism with a single chromosome set (haploid genome) of 17 chromosomes whose DNA has been completely sequenced in a collective effort of several European and U.S. laboratories (1996). This remarkable achievement makes it possible to identify new genes that regulate the life of this organism.
Yeast reproduces by budding of a daughter cell from the mother cell in a simple cell cycle, but it can also have sexual reproduction when two cells with different sexual characteristics mate after a hormonal stimulus. This diploid phase makes possible genetic analysis of mutations in genes that code for essential proteins. Yeast can be transformed (introduction of DNA in the form of a plasmid) with DNA that contains mutated genes or genes from other organisms, such as humans. It is possible in this way to study the functions of human genes in an organism that behaves at the cellular level in a manner very similar to humans.
Human genes can be mutagenized in vitro and introduced into yeast to study the effect of these mutations. Through these methods, many human genes have been characterized and structural features important to their function identified. The procedures that are used to identify and isolate mutations are called genetic screening and are generally designed to identify and isolate recessive mutations induced by treatment with mutagenic substances.
In organisms with only one chromosome set (haploids), such as Bacteria and yeast, defects caused by these induced mutations can be seen immediately. A mutation in the only available copy of a given gene will indeed have a visible effect on the cell. In diploid (double chromosome set) organisms, the mutation will be visible only if it is present on both chromosome copies (homozygosity). Genes that code for essential proteins are the most important for a genetic analysis. Expression of mutations in essential genes leads to the death of the organism, and therefore, conditional mutants must be used to study the effects of these variations.
A mutant protein may be functional at 30 °C, but completely inactive at 37 °C, while the normal protein would grow normally at both temperatures. Strains containing the mutations can be kept alive at the permissive temperature and then for genetic analysis grown at the nonpermissive temperature to study the effect of inactivation of the particular gene. An example of this type of analysis is mutations affecting the cell cycle in yeast. It is possible to follow under an optical microscope the growth and division of yeast that occurs by budding, and the size of the bud is indicative of the various stages of the cycle. At first, temperature-sensitive mutants were identified and later they were analyzed microscopically, in the search for defects in the cycle, at the non-permissive temperature.
These mutants did not grow slower than nonmutagenized cells, showing that they did not report a generic metabolic impairment, but they all stopped at a particular position in the cycle. The result showed that the mutated gene product was needed at that particular stage of the cycle. This screening allowed the isolation of CDC (Cell Division Cycle) genes that regulate the cell cycle in many organisms. The phenomenon of gene suppression is used to identify proteins that specifically interact with each other within the cell.
The principle of this analysis is based on the fact that a point mutation (replacement of a single amino acid in a protein) can induce structural changes in protein A so that it is no longer able to interact with protein B, which is involved in the same cellular process. However, mutations can occur in protein B that make it able to interact with mutated protein A.
Thus, both genes are said to be mutated, but the mutation in B suppresses the A mutation. As we have seen, genetic analysis of a simple organism can give essential information to identify the genes responsible for certain phenotypes, however, it is essential to map precisely along the chromosomes the location of these genes and then isolate them and determine their sequence. The location of a mutation in a given chromosome is the first step in mapping.
An example is recessive mutations in the X chromosome of Drosophila, but it can be extrapolated to humans. These X chromosome-related recessive mutations always show a sex-linked segregation pattern in various crosses. When crossed with normal homozygous females, males that carry the mutation produce normal offspring. All male offspring that come from a female homozygous for the mutation (on both chromosomes) will have a mutant phenotype, and all females will be heterozygous (one X from the father and one from the mother) and thus have a normal phenotype. However, the heterozygous females will act as carriers of the mutation and pass it on to 50% of the male offspring. This example shows that any recessive mutation that has a sex-linked segregation pattern can be mapped to the X chromosome.
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