The Mitochondrial genome of insects

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Minisatellites

Minisatellite DNA, additionally referred to as ‘variable number tandem repeats’ (VNTRs), is a noncoding DNA that happens at scattered sites throughout the genome (Jeffreys et al., 1985). It is composed of short (8-100 bp), tandemly recurrent motifs and also the minisatellite regions are hypervariable because of variation within the repeat number of units at every locus (Parker et al., 1998).

For a multilocus approach, genomic DNA is digestible with a frequent cutting restriction nuclease, separated by agarose gel electrophoresis, blotted to a nylon membrane and hybridized with standard repeat sequence probes. As even closely connected individuals do not share identical banding patterns, minisatellites are the marker of selection for paternity exclusion and therefore the identification of individuals. A single-locus approach by constructing locus-specific probes is mainly potential. As this method is additional labour intensive and possibly less informative than the utilization of microsatellite markers it is solely seldom used (Burke et al., 1991).

Microsatellites

Microsatellites are tandem repeated units of mono-, di-, tri-, tetra- and penta nucleotide DNA sequences, which are commonly distributed in the genome (Powell et al., 1996). Litt and Luty in 1989 used the term “microsatellites” while analysing the dispersion and abundance of (TG) n in the cardiac actin gene. Based on the repeated sequences presented microsatellites are classified as: (i) perfect, when showing only perfect repetitions, (ii) imperfect repeats, when the repeated sequence is interrupted by different nucleotides that are not repeated, and (iii) composite, when there are two or more different motifs in tandem. The composite repeats can be perfect or imperfect. The repeats of di-, tri- and tetranucleotide sequences are commonly used in molecular genetic studies (Selkoe and Toonen, 2006). Amplification of microsatellites using polymerase chain reaction (PCR) permits the amplification of single loci, thus facilitating data integration (Bravo et al., 2006). Microsatellites are widely distributed throughout the genome, highly polymorphic and transferable between species (Chistiakov et al., 2006).

Microsatellites show a high degree of length polymorphism and mutation rates vary from 10-6 to 10-2 per generation, being therefore important beyond base substitution rates in alternative elements of the genome and two to three orders of magnitude higher than values known for allozymes (Jarne and Lagoda, 1996). These high mutation rates are caused by slippage events during DNA replication, where nascent and template strand realign out of register. With the introduction of PCR and also the development of high turnout fragment analysis strategies SSRs became one in every of the foremost powerful codominant markers ever found in principle, many thousand potentially polymorphic loci ought to exist in any species (Schlotterer, 2000).

Due to the very fact that the majority of the SSRs are placed in noncoding regions with high base substitution rates, the invention of universal flanking primers applicable for larger members of taxa is not possible. Thus, microsatellite markers have to be compelled to be isolated de novo from most species to be examined for the first time (Zane et al., 2002). The normal isolation procedure consists of the development of partial genomic libraries and therefore the screening of many thousand clones by colony hybridization with SSR specific probes. Though this approach is technically easy, it is very grueling and time consuming, particularly for genomes with a low microsatellite density. Refined or various approaches for SSR isolation embody the development of enriched libraries (Ostrander et al., 1992; Chenuil et al., 2003), vectorette PCR (Lench et al., 1996), the employment of expressed sequence tags (Rungis et al., 2004) and RAPD based techniques (Lunt et al.,1999; Ender et al., 1996).

Mitochondria

Mitochondria are a unit organelles found inside most eukaryotic cells. They are observed to be little (0.5-1.0 µm), normally rod formed, with two distinct membrane bilayers encompassing them. They are responsible for the generation of cellular ATP through the oxidative phosphorylation enzyme pathway. A proton gradient over the mitochondrial inner membrane, maintained by the oxidative phosphorylation enzyme complexes, is utilised to drive the preparation of ATP from ADP and phosphate inside the mitochondrial inner matrix.

The term "mitochondria" was originally coined by Benda in 1898, formally naming these entities that had been discovered inside cells for the preceding 60 years (Ernster and Schatz, 1981). DNA was isolated from the mitochondrial organelles, showing that the mitochondria had a hereditary framework independent of the nuclear genome of the cell (Nass and Nass, 1963; Nass et al., 1965). This discovery realizes a restored eagerness within the "Serial Endosymbiotic Theory”, at first anticipated by Altmann in 1890, that the mitochondrion was the outcomes of bacteria that entered into a dependent relationship with the predecessor of the eukaryotic cell (Ernster and Schatz, 1981). Broad acknowledgement of endosymbiotic inception of mitochondria and chloroplasts appears that it may depict the theory of Gray and Doolittle (1982). Ensuing studies have supported endosymbiotic theory, and remaining discussions focusing on the Serial Endosymbiotic Theory versus a coincident origin of the mitochondria and nucleus in eukaryotic cells (Gray, 1992; Gray et al., 1999; Dyall et al., 2004a; de Grey, 2005).

Mitochondrial evolution

Current opinions of mitochondrial origin are based on molecular phylogenetic analyses of varied molecular markers that systematically describe the mitochondria as branching with extant a-proteobacteria (Viale and Arakaki, 1994; Olsen et al., 1994; Andersson et al., 1998). This endosymbiotic acquaintanceship is thought to have happened at an early phase of evolution of eukaryotes. Eukaryotic cells exists that lack mitochondria, however most groups have right away been investigated and seem to either encode genes that are clearly of mitochondrial origin, or hold organelles that will be changed mitochondria (Gray et al., 1999). Presently there are claims that organelles called hydrogenosomes is additionally to a great degree changed mitochondria that have devolved insitu of the aerobically respiring mitochondria in anaerobic eukaryotes (Hrdy et al., 2004; Boxma et al., 2005), however the proof supporting this assertion is still controversial (Dyall et al., 2004b; Gray, 2005). Mitosomes are considered going to be degenerate mitochondrial organelles (Gray et al., 2004), minimizing the potential variety of protists that will have maintained a primitive amitochondriate state (Gray, 2005).

The diversity of structural forms that existing mt-genomes have adopted is commonly not appreciated by those aware of solely animal, plant and yeast mitochondrial systems. The circular chromosome presumed to be ancestral state has been changed to single linear molecules in some protist groups, split into multiple linear chromosomes in Amoeboid protists, or developed into complicated groups of cistron coding maxicircles and minicircles that direct RNA editing in trypanosome mitochondria (Gray et al., 1999; Burger et al., 2003a; Burger et al., 2003b; Bullerwell and Gray, 2005).

Great functional diversity has conjointly been determined, as well as varied samples of RNA editing systems (Gray, 2003). There are descriptions of rRNA genes expressed as pieces that have to self-associate to function (Boer and Gray, 1988; Gillespie et al., 1999). Protein genes are divided into severally expressed protein subunits (Edqvist et al., 2000), with one portion expressed within the mitochondria and the other expressed within the cytoplasm (Nedelcu et al., 2000; Martinez et al., 2001).

The modern eukaryotic cells and their genomes are the product of a significant evolutionary process of bacterial endosymbionts transformation into permanent organelles (Gray et al., 1999). Further gene loss is due to the movement from mt-genome to the nuclear genome leading to modified nuclear co-ordination expression of the mitochondrial genes and thereby increased nuclear control over mitochondrial function (Muller and Martin, 1999; de Grey, 2005). The gene content among mt-genomes varies greatly between the various surviving groups, from 98 genes within the freshwater protozoan Reclinomonas americana (Lang et al., 1997) to solely genes in Plasmodium species (Conway et al., 2000).

Significant exertion has gone into creating a hypothesis to describe why mitochondria have actually administered any genes, and not allowed for complete transfer of genes to the nucleus. Three principle speculations are presently cited to clarify the persistence of organellar DNA; the hydrophobicity hypothesis, the codon disparity hypothesis, and location based expressional control (Adams and Palmer, 2003; de Grey, 2005). The hydrophobicity hypothesis demonstrates that mitochondrial gene products are among the foremost hydrophobic proteins, are tough to import across the outer and inner mitochondrial membranes, and argue that these properties of the proteins have prevented gene transfer to the nucleus (von Heijne, 1986; Popot and de Vitry, 1990; Claros et al., 1995). The second theory notes that the changes in genetic code between nuclear and mitochondrial genomes, particularly the common amendment of UGA to code for tryptophan in mitochondria rather than a stop codon within the nuclear code, would cause proteins being translated with severe truncations or amino acid substitutions and would be chosen against gene transfer (Andersson and Kurland, 1991; Jacobs, 1991; Leblanc et al., 1997). The sequence changes seem to possess occurred once the loss of the majority of genes therefore is also concerned in maintenance of the currently heavily reduced mt-chromosome (de Grey, 2005).

The third hypothesis proposes that key genes concerned in oxidative phosphorylation are expressed within the mitochondria in order that their proximity to the enzyme complexes might regulate their expression (Allen, 1993). Location based expressional control theory is gaining support inside the community (Race et al., 1999; Adams and Palmer, 2003; Allen, 2003; Gaspari et al., 2004). Experimental elucidation of translational-based management of mt-mRNAs in yeast (Naithani et al., 2003; Barrientos et al., 2004) and probably in humans (Mili and Roma, 2003; Mootha et al., 2003; Xu et al., 2004) are adding tidy support to the current hypothesis.

Mitochondrial genome of insects

The insect mitochondria contain double-stranded circular genomes that vary from 14,503 bp as in Mayetiola destructor (Diptera: Cecidomyiidae) (Beckenbach and Joy, 2009) to 19,517 bp in size as in D. melanogaster (Lewis et al., 1995). Inspite of the extraordinary range in size, the gene content of the molecule is remarkably conserved. It encodes 37 genes: 2 for rRNAs, 13 for proteins and 22 for tRNAs. (Song et al., 2010). Further to this, mt genome of insects holds one extensive AT-rich non-coding control elements, that are concerned within the initiation and regulation of mt transcription and replication. Hence these are referred as the mitochondrial control region (CR) or AT-rich region, which is present between srRNA and tRNA-I (Hua et al., 2008).

The entire genome except the control region consists of compactly arranged coding sequences. This region with no introns and with very few intergenic nucleotides, and neighbouring genes may even shortly overlap in few cases. Overlapping genes found around mtDNAs prompts speculation that the “polycistron model” may not generally apply, since it would not be possible to release full-length RNAs of each overlapping message from the same transcript (Montoya, 1983).

Distinction in size of the mitochondrial genome is seen because of the diverse length of the control region. For the most part the extent of the control region of mtDNA range from 70 bp in Ruspolia dubia (Zhou et al., 2007) to 4.6 kb as in Drosophila melanogaster (Lewis et al.,1995). In Hemiptera, there are available a number of completely sequenced mitochondrial genomes that have variations in the gene order (Hua et al., 2008; Song and Liang, 2009). The observed rearrangements fall under 3 types: (A) translocations and transpositions of tRNA genes on the basis of the ancestral gene order; (B) I-Q-M-ND2-W-C-Y block transposed to the end of the control region, with the translocation of some tRNA genes; (C) transpositions of DNA section containing COIII-G-ND3-A-R-N on the basis of type B. In type C, there is variation within the mitochondrial position into that these genes are transposed. Furthermore, there are differences the maintenance of the number and the order of the excised tRNA genes at the mitochondrial area in which the genes are embedded.

The maximal insertion includes majority of the genes from the excised fragment in their original order A-R-N-ND3-G-COIII, the least insertion involves ND3-G-COIII. In all insertions, the transcription direction is changed from that within the original position (Thao et al., 2004). Mitochondrial genome sequences are, no doubt decided at an increasing rate, providing several new opportunities for comparing genome structure. MtDNA data have assumed a significant part in investigations of molecular evolution that have helped to refine models of evolution.

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