Role of symbiotic bacteria in pseudo germline transformation of Drosophila melanogaster with the Pip1/piggyBac construct by crossing studies

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Analysis of the role of symbiotic bacteria in pseudo germline transformation of Drosophila melanogaster with the Pip1/piggyBac construct by crossing studies


  1. Transposable elements

Barbara McClintock’s observation of the variation in kernel phenotypes led to the discovery of the transposable elements (TE’s) which changed the way of how scientists thought about genetic patterns of inheritance (Pray & Zhaurova, 2008). TE’s, also called jumping genes, are DNA sequences that move around to different positions within the genome of a single cell by a process called transposition (Malvee, 2010). During this process they can change the amount of DNA in the genome and cause mutations. Their abundance and diversity have contributed to the differences in the structure and the size of eukaryotic genome (Gilbert, 2011). Transposons or TE’s are normally grouped into two classes. The class I transposons (RNA-mediated) follow a copy and paste method by which they copy themselves by initially being transcribed to RNA, then to DNA (by reverse transcriptase) and finally they get inserted at another position in the genome (Malvee, 2010). Class I TE’s are further divided into long terminal repeat (LTR) retrotransposons, non-LTR retroposons or LINES’s (long interspersed nuclear elements) and SINES’s (short interspersed nuclear elements) (Gilbert, 2011). SINES have the highest copy number (in terms of interspersed repetitive DNA) in the human genome with over 1.7 million copies, comprising 14% of the genome as a whole whereas LINES make up over 20% (Brown, 2007). SINE’s cannot move to new locations in the genome, they require the LINE reverse transcriptase and an endonuclease protein for its movement (Clark & Pazdernik, 2012). Most of the retroposons contain two open reading frames (ORF). ORF1 encodes a nucleic acid binding protein and ORF2 encodes reverse transcriptase and the endonuclease (apurinic/apyrimidic) (López & Pérez, 2010). The integration of a retroposon to a new place is an irreversible event (Bilnov, Glushkov & Novikova, 2006). Some examples of the retroposons are the Juan C, Juan A and the Pip 1 element. Retroposons can be around 3-8 kilobases long and most of them have a pol-like protein that includes a reverse transcriptase domain necessary for retrotransposition (Gilbert, 2011).

Class II transposons (DNA mediated) move directly from one position to another by following a cut and paste mechanism. They transpose directly from DNA to DNA and no RNA intermediate is involved in the process (Gilbert, 2011). They work in different ways; some can bind to any part of the DNA molecule and the target site while others bind to specific sequences (Malvee, 2010). Transposons are considered powerful molecular genetic tools for performing genome wide analysis and studies related to a particular gene or the gene region. There are many different transposon systems used as mutagenesis tools (Bellen & Venken, 2007). For each type of transposon there is an element specific protein which is known as a transposase. A transposase catalyzes the biochemical steps involved in transposition. In addition to the presence of a transposase, some transposons also require host proteins or other additional transposon encoded proteins (Schaechter, 2009).

  1. Transposable elements in Drosophila melanogaster – use in germline transformation

In Drosophila many of the spontaneous mutations and the rearrangements in the chromosomes are known to be caused by TE’s, examples which are the P elements, Piggybac and the copia- like elements. Around 10% of the Drosophila genome consists of these repetitive DNA (Gelbart, Griffiths, Lewontin & Miller, 1999).

P elements are highly mobile TE’s that were first isolated in 1982 from Drosophila melanogaster (Handler, Gomez & O’Brochta, 1993). The mobility properties of this TE allow its development into an efficient gene vector thereby making it one of the important tools used in the study of Drosophila genetics. Approximately 30-50 copies of the P element were found throughout the arms in the fly genome (Handler et al. 1993). The P element is the factor responsible for hybrid dysgenesis that occurred in crosses of males from a P (paternal) strain with females from M (lacking P factor) strain (Bingham, Kidwell & Rubin, 1982). It follows a cut and paste method and functions as a vehicle for insertional mutagenesis elements. The P elements transposes in the germ line cells only due to their alternate splicing structure. Mobility assays that could test transposon function in embryos proved that the P element did not function outside the Drosophilidae (Handler et al., 1993) hence the search for a new transposable element had begun and Piggybac transposon was discovered. The PiggyBac (PB) TE was discovered in a moth Trichoplusia ni, originally isolated from a baculovirus (Rohrmann, 2013). It is currently the preferred choice of vector for gene discovery and identifying gene function in many insects and mammals because of its mobility in their genomes (Cheng, Duan, Liu, Xia, Xu, Zha & Zhao, 2006). PB transposes between chromosomes and vectors using the cut and paste method. It encodes a 2.1 kb transcript consisting of a single ORF which is 1.8 kb in length (Cheng et al., 2006). The PB transposase is flanked by two 13 basepairs inverted terminal sequences and is 2472 bp long (Cary, Goebel, Corsaro, Wang, Rosen & Fraser, 1989). During their transposition the PB transposase recognizes transposon specific inverted terminal repeat (ITR) sequences which are located at both ends of the transposon vector. It then helps in moving the contents from the original sites and integrating them into the TTAA chromosomal sites (Cary et al., 1989). The two terminal repeats- 5’TR and 3’TR also help to identify the orientation during integration. This advantage of the PB transposase to remain active when fused with other proteins opens a wide range of possibilities for temporally controlled mutagenesis. Some of the Piggybac transposons are reversible too. Both P element and the PB transposons are used for the germline transformation of Drosophila and many other insects (Handler, 2002). The germline transformation of the Drosophila melanogaster strains had been carried out with the PB transposable system in this experiment. Microinjecting foreign DNA into embryos with a mixture of two plasmids, one that encodes a transposase and the other with the ends of the transposon containing the gene, is termed as germline transformation (Verma & Singh, 2014). The DNA to be injected involves two components, the helper plasmid which although can produce PB transposase, is itself immobile and the other component consists of a transposon construct in which the sequence to be integrated as a transgene is situated between the PB element ITR (Chia & Connor, 2002). These transposons act as a tool for germline transformation by integrating into the plasmid vectors after which they undergo further modifications. PB has been used to transform the germline of more than a dozen species. Their nature of transposition helps in genetic analysis and insertional mutagenesis projects (Handler, 2002).

  1. Symbiotic relation of wolbachia and Drosophila melanogaster.

Just like humans, many insects have a symbiotic relation with the bacteria. Symbiosis can be defined as being “living together of two completely different organisms” (Steffen, 1982). A symbiotic association with the host offers many advantages. One such example is the wolbachia, an α-Protobacteria that infect many insects (Ashburner, Ferreira & Teixeira, 2008) and is known to cause parthenogenesis (offsprings developed from unfertilized eggs), male killing, feminization and cytoplasmic incompatibility (when infected males breed with non-infected females)(Benzer & Min, 1997). Wolbachia, an organism from the Rickettsial family, resides in the cytoplasm of the host cells and are mainly known for disrupting the reproductive biology of their hosts to increase their transmission through the female germline (Ashburner, Ferreira & Teixeira, 2008). They were first discovered in Culex pipiens, a blood feeding mosquito. Wolbachia strains can be classified on the basis of their cytoplasmic incompatibility or using Wolbachia gene sequences which is 16S rDNA. 16S rDNA is a part of prokaryotic DNA. The ‘r’ stands for ribosomal and codes for a strand of RNA that is a part of the ribosome. This gene is considered useful since it is quite short as compared to the other genes that are found in bacteria. Wolbachia are detected in a large proportion of Drosophila melanogaster, especially within the laboratory stocks (Anderson, Cande, Clark & Karr, 2005) and are carried in a non-virulent form which affects their behaviour and longevity (Benzer & Min, 1997). Most of the reports state low levels of CI in Drosophila melanogaster, CI is dependent on many factors like age of males, wolbachia strain, the environmental conditions and its levels may vary because of the density of bacteria carried by the insects (Dagher, Hercus & Hoffman, 1998) Different laboratory studies have shown that Wolbachia decreases or has a little impact on fecundity, i.e., the actual reproductive rate of an organism measured by the number of eggs (Hoffman 1990) but it may increase fecundity under appropriate environmental conditions (Hoffman 2000).

The fruit flies provided in this experiment had full length copies of Pip1 and JuanC being inserted into the PB vector and injected into the embryo of Drosophila melanogaster using a helper plasmid. Therefore, Pip 1 and Juan C construct are plasmids consisting of the PB ITR at both ends which is approximately 30 bp. Both Pip and Juan-C are jockey clade non LTR retrotransposon. In Drosophila melanogaster, many clades have been identified, one of which is the jockey clade containing several subfamilies (Berezikov, Bucheton & Busseau, 2000). The pip 1 and Juan C were integrated into the germline of the Drosophila melanogaster. It was hypothesised that the Pip plasmid is present in the symbiotic bacteria rather than in the genome of Drosophila melanogaster. This was tested by looking for non-reciprocal inheritance of the plasmid in crosses to wild type flies. The strains used were canton S (wild type strain), Yellow white. The yellow white strains were germ-line transformed with the Pip1 and Juan C retroposon individually. Crosses between Canton S x Pip (including the reciprocal cross) was carried out, in order to find the presence of Pip1 plasmid in the offspring’s. The presence of Pip1 plasmid was confirmed through PCR.


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