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Genetic Tools In D. Melanogaster And It'S Gal4-Uas System

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Genetic tools in D. melanogaster

Balancer chromosomes

One of the conveniences afforded in D. melanogaster studies is the availability of balancer chromosomes (Hochman, 1971). They are useful for a number of reasons. For one, many of the more interesting genes to study in D. melanogaster are recessive lethal; as a consequence, the maintenance of the mutation within the population relies on heterozygotes. This situation is less than desirable, as the recessive nature of these genes also means that heterozygotes could not be selected through simple phenotype screening. Additionally, as heterozygotes are crossed to one another and crossing over occurs, the mutant allele will slowly be weeded out through the loss of recessive homozygotes, and thus the mutation will eventually disappear.

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Balancer chromosomes come into play as constructs into which multiple nested inversions along with at least one dominant marker are introduced. These chromosomal mutations are important, as they simultaneously suppress the resulting chromosome from undergoing homologous recombination and allow for heterozygotes to be easily picked out from the offspring population. When two or more recessive lethal mutations are to be investigated and lay in cis to one another on the same chromosome, the balancer system eliminates the need to select for heterozygotes completely, as by principle of lethality no offspring homozygous for either mutation will survive (Mason, Ransom, & Konev, 2004; Zheng et al., 1999). In D. melanogaster, balancers for X chromosomes and autosomes 2 and 3 have been widely used to propagate and maintain lethal mutations in a line over generations. They will be seen in several lines under investigation for this study, and the specific subset of dominant phenotypes each balancer carries will facilitate selection during screening.

GAL4-UAS system

Another advantage in having D. melanogaster as a model organism is the ability to utilize its GAL4-UAS system. The biochemical method has proven to be a powerful tool in screening, ever since it was first described to be sufficiently robust for application in fly genetics (Brand & Perrimon, 1993).

GAL4 is a transcription activator protein originating from yeast. It consists of two protein modules: an activation domain and a DNA-binding domain. Through the latter, GAL4 recognizes and subsequently binds with high affinity to CGG-N11-CCG of the Upstream Activation Sequence (UAS) (Campbell et al., 2008). In this binary system, UAS is the expression vector, and is typically constructed such that it precedes a cloned gene of interest. When GAL4 proteins are expressed where UAS sites are also found within the cell, concurrent expression of the attached gene and the effects its expression would confer unto the cell system could make way for the elucidation of the gene’s molecular functions. Through traditional molecular cloning methods utilizing transposition-enabled vectors followed by embryo injection and selection of transgenic individuals, these genes are introduced into the genome of the fly (Brand & Perrimon, 1993). The GAL4-UAS system has proved so effective that thousands of lines have been generated, all of which are readily available upon request from any one of three major stock deposition centres (Bloomington, Kyoto, and Vienna).

This extensive availability provides avenues for the researcher to tailor crosses to involve only tissue-specific drivers or promoters in directing the expression of GAL4 in D. melanogaster. The construct is manipulated such that GAL4 protein is only expressed in a particular tissue. Non-drosophilid origins of GAL4-UAS immensely assists cell-specific genetic screening in this case, as none but cells where the expression of the driving promoter is endogenously active would exhibit changes. A second line of flies is then generated carrying the construct for UAS along with the gene of interest (Phelps & Brand, 1998). As neither GAL4 nor UAS have any effect on their own, following crossing, only progenies should display the consequence of the gene’s activation.

Oftentimes the UAS construct includes a reporter gene such as the Green Fluorescent Protein (GFP). This allows selection of progenies expressing the gene of interest through a surface-phenotype under ultraviolet light, without the need for downstream antibody staining and visualization steps. Driver-GAL4-UAS-reporter systems sans any other cloned genes have also been used to great effect in studies into temporal-specific neuronal migration patterns (Morante, Erclik, & Desplan, 2011) as well as ion-transport across certain cell types (Ng, Tangredi, & Jackson, 2011).

Further applications of GAL4-UAS: MARCM

Balancer chromosomes enable the sustenance of otherwise lethal alleles in a population. However, comparative expression studies between heterozygotes and homozygotes would not be possible, as those in the latter group do not survive past the larval stages. In this case, it is useful to study genetically mosaic individuals, whereby clones of somatic tissues could have a varying genotype when compared to the others within the same body. This ensures that while the individual itself remains viable due to its largely heterozygote genotype, at a desired point of development a cell can be induced to mitotically recombine in order to produce a homozygote (Hall, 1988). Descendants of this cell, and only this cell, will be homozygotes, and comparisons between them and immediately neighbouring heterozygotes is not only made visually convenient, but the applications of this principle has facilitated much of research into gene functions.

A specially developed mosaic system in D. melanogaster named the Mosaic Analysis with a Repressible Cell Marker or ‘MARCM’ technique (Lee & Luo, 1999, 2001) is developed through the utilization of the GAL4-UAS system alongside Flp/FRT recombination. It has proven to be indispensable in enhancing our understanding of the organism, especially in terms of neuronal cell characterization.

Flp/FRT recombination is yet another S. cerevisiae derived genetic manipulation tool (Golic & Lindquist, 1989). Flp or flippase is a recombinase, and Flippase-Recognition Target (FRT) is its binding site. In the heterozygous progenitor, FLP is controlled by an inducible promoter. On another chromosome, GAL80 – a gene encoding a natural inhibitor of GAL4 – and a gene or mutation of interest is arranged in trans to one another, and each distal to an FRT site. The system also includes a visible marker such as GFP (Xu & Rubin, 1993), whose expression is dependent upon a UAS promoter. GAL4 is under the control of a ubiquitous promoter, but its UAS-activating activity is repressed by the presence of GAL80. These elements combined create a GFP-negative cell.

When the promoter preceding the FLP gene is induced, Flp catalyzes mitotic recombination of the regions distal to FRT. Once mitosis is complete, this creates two homozygotic progenitor cells: one for GAL80, and another for the gene or mutation under study. This second homozygote is free of GAL80 proteins, enabling GAL4 to activate UAS. All descendant cells will thus be GFP-positive and by focusing on them, the effects of the gene or mutation could then be analysed (Wu & Luo, 2006).

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