Results and discussion

analysis can be used to localize the cis-regulatory regions of a gene (Kipp and Mayo, 2009; Yavatkar et al., 2008). Intensive studies and computational approaches have been undertaken in dissecting human c-myc promoter, as comprehensively summarized in Wierstra (2008). The studies have resulted in identification of a variety of repressor and activator binding sites in the enhancer regions throughout the gene body. The investigations have further shown that the c-myc gene regulatory region is modularly structured; it contains four promoter (P1, P2, P3, and P0) and two polyadenylation sites (Marcu et al., 1992; Spencer and Groudine, 1991). However, it has been very difficult to characterize enhancer elements responsible for correct expression of c-myc gene. In one of the in vivo studies Mautner et al. (1996) tested constructs including up to 50-kb of the c-myc locus, but the constructs failed to express exogenous c-myc gene substantially. The insertion of the immunoglobulin κ-intron and 3’ enhancers, however, activates c-myc transcription when placed adjacent to or separated from the c-myc promoters by as much as 30-kb (Mautner et

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al., 1996). The coupling of these enhancers with the c-myc gene mimics the chromosomal translocation in Burkitt’s lymphoma, in which the c-myc gene gets translocated to the immunoglobulin loci. Although the distance of the chromosomal breakpoints from the c-myc locus varies from 1-kb to 350-kb, the translocated c-myc becomes actively transcribed from the P1 promoter.

The above findings indicate that the examined sequences of 50-kb contain functional promoter elements but lack at least some essential enhancer elements. Given the large size of 160 to 350-kb, the identification of additional enhancer elements in the c-myc region is expected to be difficult (Gombert et al., 2003; Mautner et al., 1996).

Such studies can more efficiently and conveniently be performed by investigating myc regulation in the fruit fly for several reasons: (i) Drosophila possesses well-established genetic tools; (ii) dmyc is the sole homolog of c-myc in the fly; (iii) dmyc locus spans over 40 to 50-kb, instead of 160 to 350-kb, (iv) life cycle of Drosophila is much shorter than mouse life cycle.

1.6.1 Computational Approaches

The occurrence of conserved regions and repetitive sequence motifs in noncoding DNA has been of great value for the identification and characterization of cis-regulatory elements. The phylogenetic footprinting tools EvoPrinter (Yavatkar et al., 2008) and cis-Decoder (Brody et al., 2007) are efficient software tools that can readily serve for the identification of conserved sequence blocks in developmental genes. EvoPrinter facilitates the multialignment and rapid identification of evolutionarily conserved sequence blocks as they exist in the species of interest. The cis-Decoder then characterizes repeat motifs within the conserved sequence blocks and detects conserved elements among functionally related enhancers. It is important to mention that EvoPrinter and cis-Decoder do not detect polyadenylation signals or core promoter elements, such as TATA boxes.

The neural network genetic algorithm PROMOTER 2.0 can be used to predict promoter region in eukaryotic genes (Knudsen, 1999). PROMOTER 2.0, combined with the consideration of CCAAT or bHLH recognition motifs facilitates the recognition of discrete subpatterns that are recognized by RNA Pol II. The identified subpatterns can then be further analyzed by the bioinformatics tool DNASTAR laser Gene, module GeneQuest, to detect cis-acting transcriptional regulatory elements

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such as GC box, 5’-GCGCGGC-3’ (transcription factor SP1 binding site) (Song et al., 2009), TATA box (also called Goldberg-Hogness box) (Lifton et al., 1978), Initiator Element (transcription initiation site) (Smale et al., 1998; Smale and Kadonaga, 2003), Downstream Promoter Element (DPE) (Smale, 2001) and transcription termination signals in the 3’-UTR region.

The data obtained by computational methods serve as a supportive tool for the generation of overlapping deletion constructs covering the entire noncoding regions of a gene that can be used to perform reporter activity studies.

1.6.2 Reporter Activity Study

Reporter gene assay is an effective and easy method for the measurement of the activity of a particular promoter in vivo. A common reporter is E. coli lacZ gene, which resides within the lac operon and encodes the β-galactosidase (β-gal) enzyme. The enzyme is 120 kDa and for its activity forms a tetramer. In bacteria the enzyme cleaves lactose to glucose and galactose to provide the cell with the source for carbon and energy.

In vitro the synthetic compound 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-gal) consists of galactose linked to an indole molecule; it can be cleaved by the enzyme β-gal. The insoluble cleavage product, an indoxyl glycoside has a blue color and is indicative for the presence of β-gal enzyme (Kiernan, 2007). The accurate detection of the enzyme β-gal in the system allows the use of lacZ reporter as a measurement for promoter activity.

Two major transgenesis approaches for the study of reporter activity in Drosophila melanogaster have been established, namely random P-element transformation (Rubin and Spradling, 1983) and site-directed phage ΦC31 integrase transgenesis system (Bischof et al., 2007; Venken et al., 2006). On the basis of computational analyses of the noncoding regions of a gene, both transgenesis systems can be used to introduce different truncations of the noncoding regions of a gene into the fly genome and study the expression of a reporter under control of a particular promoter.

Random P-element transformation requires analysis of many independent lines to avoid insertion locus position effect on the promoter activity. Site directed mutagenesis takes advantage from the phage attachment sites attB and attP, and

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allows the irreversible integration of a transgene into a particular landing site in the genome. This approach helps to save extra labor and avoids position variegation effects. Since the method is new in the field of reporter activity studies, a combination of both methods ensures more precise interpretation of the promoter activity.