Zebrafish (Danio rerio) are the small fish under the group of cyrinide from member of telosts and present in the freshwater origin from India and Burma. It grows adults length upto 50mm and life span nearly 3 years. Female fish lays the eggs approximately 100-200 eggs per week and can be fertilized externally and develop together. In modern scientific research field, Zebrafish are play an important role in developmental biology. Zebrafish embryos are very transparent and develop very quickly and it can be observe greatly in developmental processs and imaging. (McGonnell and Fowkes, 2006). Zebrafish mitochondrial genome structure consist of 165966p and contains 13 protein coding gene, 7 subunits of NADH(uniquinone oxidoruductase), 1 subunits of cytochrome C(cytb), 3 subunits of oxidase complex and 2 ATP synthase and ribosomal DNA contains small subunits(12S) and large subunits(large subunits(16S) and tRNA gene(22). A noncoding region has contain to initiate light and heavy strand replication which present inside 5 tRNA genes. (Howley C and Ho RK, 2000).
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In female, Oogenesis or egg formation takes place in ovary. Each ovary contain diploid cells called oogonia to right from embryonic germ cell. Before birth oogonia divided by mitosis. And some of them develop in primary oocyte. Primary oocyte contain immature egg cell contain with massive cells called follicle. (Fleming et al,.(1983).Primary oocytes then inner meiosis 1 process stop uncompleted and their puberty. No primary oocytes for act in this point. At birth each female has the finite number of primary oocytes available for reproduction. At puberty complex form to stimulate change in the follicle and give the some primary oocytes complete their mitosis1 activation. The divitional cytoplasm and the cell organell is unequal as result one large secondary oocytes and one small polar bodies formed per primary oocytes. Eppig J (2010). The polar body after degenerates the follicle contain secondary oocytes continued material and serve to move the edge. Initiate ovulation the mature follicle repture releasing the secondary oocytes at this point secondary oocytes start meiosis 2 begins. This meiosis 2 will not completed over all unless fertilization occur. The secondary oocytes envelope the oviduct where fertilization of the oocytes with sperm cell can occur. The entry of the sperm cell into the cytoplasm of the secondary oocyte strickes the completion of the meiosis 2. Cytoplasm divide unequally generate the mature ovum and secondary polar body. The fusion of the haploid sperm cell and haploid egg cell has product the diploid zygote cell.( Spikings, 2007).
Structure of an Oovary ( Ova production, 2010).
Ooctyes development in zebrafish consists of 5 stages depends on morphological and biochemical basis. During Primary growth stage (stage1), oocytes consist of no yolk and looking like a transparent in cytoplasm which is sourrounded by germinal vesicles. In cortical alveolus stage (stage2), the oocytes are differentiated by appearance of different sized cortical alveoli and can be seen easily in vitelline envelope. During stage 3(vitellogenesis), it involves the collection of lipids and yolk bodies present in the oocytes. Oocytes maturation stage(stage 4) Involves the oocytes increase in size and appear translucent. Yolks are looking like non-crystalline which admitted meiotic maturation. During mature egg stage (stage 5) the eggs are approximately 0.75mm in size. Howley C and Ho RK (2000).
Different stages of oogenesis and egg activation from (Pelegri F, 2003).
Oocytes are ovulated in the ovarian lumen and now it can be capable of fertilization. The collections of maternal products are involved in the process of early stage of ovarian follicle development. At this development, the nucleus cell are gathered at the boundary of the nucleus and it is mainly involving the replication of ribosomal RNA. After this stage, follicle development are condensed the chromosome and extends the DNA structure which contain proteins and RNA. (Selman et al,.(1993). For transcription and replication process, RNA and protein are play an pivotal role in development stage. So isolating mRNA from ovarian follicle are very useful in expression of gene in mitochondrial DNA replication and transcription.
Mitochondria are the closed double membrane and rod-like structure present in the eukaryotic organells. The Mitochondria membrance structure consists of outer membrane and inner-membrane components and in between these components, inner-membrane space is present. The outermembrane is made up of phospholipids and protein mainly functioning as the permeable to ions, ATP and ADP molecules. Inner membrane consists of fold like structure called cristae. Cristae are the place or process of electron transport chain and it generate the ATP production by oxidative phosporylation. This aerobic respiration process are involved in another metabolism like anaerobic glygolysis and citric acid cycle and their end products are carbon dioxide and water. It release 32 molecules of ATP in aerobic respiration and anerobic respiration generates about 2ATP molecules. (Oliveira et al,. 2007). By addition of mitochondria are included the urea cycle, fatty acid oxidation, iron-sulfar cluster, lipid, amino acid and nucleotide and heme biosynthesis. Lemires B (2005).
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Mitochondria are play a pivotal role in reactive oxygen spices to actively involved in senescence and premature. Mitochondria are the power house of the cell contain molecular machinery for the conversion energy from the breakdown of glucose into ATP, the energy currency of the cell. In the two membrane, inner membrane is fold into cristae, provided large space to produces enzymatic process occurs. the inner matrix contain enzymatic system creb's cycle which contain mitochondria their own DNA genome and pure identical molecular double standard DNA and all mitochondrial DNA is inheritance from mother and other components reproduce by dividing. The biological cell tells the mitochondrial are descented from bacteria where corporate into the cell in the symbiotic cell.(Iborra et al,. 2004).
Structure of Mitochondria from (Matthew Damstrom Organelles project, 2010).
Mitochondria are the tiny packed structure present with the cells that can be convert the energy from food to the cell. Actually DNA is made up of condensed chromosomes inside the nucleus and same mitochondria also have the small amount of the DNA molecules. This genetic components is called mitochondrial DNA. In the cell, the mitochondria are present in more than thousand of amount and it is present in DNA circular shape away from bacterial ancestry. (Mitochondria: structure and Function, 2010).
Mitochondrial gene are accumulated in the mitochondria chromosome which passes one generation to the another generation in most mammals and that inheritance in maternal inheritance is reason being DNA mitochondria in it. The sperm mitochondria lost when sperm fertilized the eggs. So even though the zygote carrier the nucleus DNA from the mother cells and essential these mitochondria chromosomes passes one generation to next generation it behind to divert not because cumulative mutation. (Shourbagy et al,. 2006). Mitochondrial genome which contain double standard circular DNA about 16.5Kb with differing the size between species to species like human contain 165696bp, the pig contains 16679bp and the mouse contains 162956bp. Nucleic DNA is slightly different from genetic code to mitochondrial DNA. (Spikings, 2007). Zebrafish mitochondrial genome structure consist of 165966p and contains 13 protein coding gene, 7 subunits of NADH(uniquinone oxidoruductase), 1 subunits of cytochrome C(cytb), 3 subunits of oxidase complex and 2 ATP synthase and ribosomal DNA contains small subunits(12S) and large subunits(large subunits(16S) and tRNA gene(22). A noncoding region has contain to initiate light and heavy strand replication which present inside of the 5 tRNA genes. Howley C and Ho RK (2000).
The Mitochondrial genome (Barcoding Life, 2010)
Mitochondrial DNA are mainly responsible for somatic mutation (non-inheritance). Somatic mutation cause the damages in DNA structure and perform same alteration and deletion in DNA sequence. In respect of this, not passes the functional activity from one generation to future generation. Somatic mutation generates the potentially virulent molecules in mitochondrial DNA is called reactive oxygen species (ROS). So it can be easily causes harmful to the mitochondrial DNA. Mitochondrail DNA (2009). In mitochondria, more than thousands of mitochondrial DNA are passed their generation by maternally completed in cytoplasm of oocytes. If the zygote can receive in addition of more than one mitochondrial DNA and it will gives the different form of cell proliferation and cell division. This causes the different cell lineages and forms the mutation. So mitochondrial are play an important role in the cell physiology and somatic mutation or inherited mutation mainly involving the developmental abnormalities. The high degree level of mutation in mitochondrial DNA molecules causes the damages during embryogenesis. (Broughton et al,. 2001).
Mitochondrial DNA replication and Transcription
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mtDNA replication can occur at any phase of cell cycle and hence implies that they are independent of the nuclear DNA replication. In a metabolically active cell majority of the mtDNA exists as a triple stranded structure called the displacement loop (D-loop). The D-loop is a nacent H-strand DNA segment of 500-700 nt that remains annealed to the L-strand, this constitutes the major non-coding segment, containing main regulatory elements for mtDNA transcription and replication. The H-strand encodes most of the information: 14 of the 22 tRNA and rRNA-encoding genes and the mRNAs for 12 out of 13 polypeptides. The L-stand encodes remaining eight tRNA and one mRNA (Anderson et al. 1981). The gene products of these mRNA are essential for the function of the mitochondria. The major difference between the mtDNA and the nuclear DNA is the compact gene organization where in all the coding sequences are contiguous to each other separated by a few bases and without introns (Zeviani et al. 1998).
There are only two non-coding regions in the mtDNA which play an important role in DNA replication. One is the D-loop region situated between genes that encode tRNA for phenylalanine and proline. This region contains the origin of replication for the H-strand (OH) and the promoters for the H- and L-stand transcription named PH and PL respectively. The second non-coding region is a much shorter segment of DNA which is roughly 30 nucleotides long. This region contains the origin of replication for the L-strand (OL). This region is located inside a tRNA cluster at about two-third of the length from the OH. the strands of the mtDNA can be distinguished by their difference in G+T content and can be separated based on their density in denaturing gradient giving rise to Heavy or H-strand and light or L-strand.
The transcription from both the PH and PL promoters proceed through the entire mtDNA and results in a polycistronic RNA. The transcripts contain tRNA genes between the larger rRNA and mRNA sequences. The folding of tRNA aids in the cleaving of the mRNA and rRNA from its polycistronic transcript. These mRNAs are later post-transcriptionally modified by polyadenylation and the tRNA by addition of 3' terminal CCA (Taanman 1999).
Transcription and replication of mitochondrial DNA works under the nucleus-encoded protein inside the region of the D-loop displacement. In the process of transcription required the elements like Mitochondrial RNA polymerase, TFAM Aand TMB1M or TMB2M .Replication of Mitochondrial DNA is mainly involved in nuclear-encoded protein, especially mitochondrial DNA specific polymerase gamma. Polymerase gamma are consists of two subunits, they are POLGA and POLGB. POLGA are catalytic subnit which is subjected to elaongation of new daughter stands and POLGB are accessory subunits which is involved to primer identity and proof reading process. . The primer for initiation of DNA replication involved by procedure of light strand transcripts with the Mitochondrial RNA Processing. Thus the requirement of the transcript to produce primer highlights the inter-relationship between transcription and the mtDNA replication.
Mitochondrial DNA Replication and Transcription taken from the picture (Spikings, 2007)
Mitochondrial DNA replication process has two mechanism. First one is asynchronous displacement replicaion mechanism and second one is leading and lagging strand mechanism.The replication of mtDNA is initiated at OH using the above mentioned primer and proceeds displacing the parental H strand. The enlargement of this displacement loop (D-loop) towards the OL region is two third of the length of the mtDNA. The displacement of the H-strand exposes the OL region which forms a stem loop structure leading to the initiation of L-strand synthesis which proceeds back along the exposed single stranded H-strand template termed Exp-D. This is called as the asynchronous-displacement mechanism (Clayton, 1982). This asymmetric synthesis leads to a incompletely synthesis of the L-strand called the gapped circle (GpC) and it is segregated prior to completion of replication.
Recently another mechanism of mtDNA replication has been proposed called the leading and lagging strand mechanism by Holt et al (2000). In this model, both the strands of mtDNA is said to be synthesized bidirectionally and the double stranded replication fork proceeds through the length of the mtDNA. Here zone of replication is believed to be present beyond the D-loop. Exp-D and GpC forms are excluded by this mode of replication. (Spikings, 2007)
Polymerase Gamma (PoL Î»)
Mitochondria DNA is polymerized by mitochondrial specific DNA polymerase gamma, (DNApolï»» or PolG) and belongs to the heterotrimeric enzyme. The enzyme consists of two subunits, the large catalytic Î± subunit of 125-140 kDa and the smaller Î² subunit of 30-54 kDa. The Î± subunit (catalytic subunit) is responsible for elongation of the daughter strands where as the Î² subunit is essential for proof reading and primer recognition. Î² subunit(accessory units) is called the repair enzyme which actively involved repair mechanism of the polymerase. (Oliveira et al,.2007). In mitochondria DNA, DNA polymerase Î² are mainly started the elongation process in region of D-displacement loop.Apart from the DNA polymerase replication is also dependent on other proteins like mitochondrial helicase. mitochondrial single stranded binding protein and DNA primase involved.( Spikings, 2007). DNA polymerase gamma are home of place for the mitochondrial disease around 100 disease occurred by mutation and disease like Neuropathy, syndrome, Alpers syndrome, midlife-onset ataxia and male infertility. so we can easily figure out the mechanism of mutation by which genes caused the imbalance of mitochondrial DNA. So DNA polymersase Î³ are play an important role in replication of mitochondria DNA and If any changes or mutation occur in the catalytic subunits will cause mitochondrial disorder. So we can analyse these genes with extensive detecting techniques like RT-PCR, Microarray and Northern blotting. Chan and Copeland,2009)
TFAM( Mitochondrail transcription factor A) are type of protein belongs to HMG (high mobility group ) membrane and is mainly involved the mitochondri DNA maintainance. It has 2 HMG box-domain. HMG protein are respondible for wrapping, unwinding DNA sequence and binding. (Alam et al,. 2003) In mitochondrial DNA, TFAM is play an pivotal role in mechanism of replication and transcription. When TFAM levels are increased within mitochondria, it will increase the level of mitochondrial DNA replication and mitochondrial DNA transcription. It is very important for embryonic development. When TFAM levels are decrease, it induces the variety of mitochondrial disorders like familial mitochondrial disorders like familial mtDNA liver disease, infantile mitochondrial myopathy, skeletal muscle, fatal childhood myopathy, mitochondrial encephalomyopathy disease, muscle wasting and ocular myopathy. And also reducing the TFAM expression in mitochondria when spermatogenesis in mammals. This will reduces the mtDNA copy number and decrease the transmission of paternal mtDNA to one generation to future generation.( Spikings, 2007).
House keeping Gene
House keeping gene are the type of gene which is present in all tissue and maintain the cellular function. These genes are only present in the particular organ or tissues and involving the particular development function and cellular activity. Gene expression is very important for analysis normal and abnormal biological condition. Comparing between housekeeping gene and particular tissue gene are very essential. So these genes are play an important role to analysis the normal functional condition of particular tissues. For expression of gene, there are so may techniques used for biological research, clinical use and diagnosis main techniques like RT- PCR, Northern blotting, microarray and western blotting. The most familiar house keeping genes are Î²-actin, Î²-tubulin, GAPDH and EF1Î± (Ferrari, 2005). Actin is the globular protein present mostly in the eukaryotic cell. Actin has two types of filaments namely cytoskeleton and microfilaments present in the muscle cell. EF1Î± is the gene that responsible for elongation of aminoacyt tRNA to the palce of ribosome. Isoform (alpha 1) are present in placenta, kindney and liver. Isoform (alpha 2) are presented in heart and brain. EFA1 Î± are mostly presented multiple copies in chromosomes. (NCBI,2010) These two housekeeping genes are play an important role in mitochondrial DNA replication and transcription.
Real Time PCR
Real time PCR is the modern technoique and highly flexible to investicate, amplify, detect, manipulate and analyses from different source. Real time PCR are fluorescence technique labeling the double standard DNA sequence by using DNA intercalating dye and Sybr green. Traditional PCR are detecting the amplifier DNA at the end of the reaction. It is not giving the proper result. But real time PCR are very sensitive and detect the large quantity of amplicon at the time of reaction. (Newton and Graham, 1994 ). Real timePCR is the quantitative technique to measure the amount of mRNA or complementary DNA in the given sample from tissue or single use. Real time PCR are mainly used to measure the gene expression of mRNA at certain condition. Real-time PCR,(2008).
For this study, we are using Zebra fish and will extract the ovarian follicle at different stages of development and assess their viability and analysis with fluorescent spectroscopy. From this we will extract the RNA and it will convert onto cDNA by reverse transcription. After that we will use real time PCR and will expresses the TFAM, POLGA and POLGB genes.
Methodology of this proposal is
Collection of ovarian follicle from zebra fish
Check the viability of follicle using Trypan Blue viabible test
Convert RNA to cDNA using reverse transcription
Apply in real time PCR
Expression of TFAM, PolGA and PolGB
Aim and Hypothesis
The Proposal aim is to determine the mitochondrial DNA factor and to measure the expression of TFAM, POLGA and POLGB and compare with housekeeping gene Î²-actin and EF1 Î± by using the model of Zebrafish ovarian follicle and with the help of the real time PCR. From this study we will analyses mitochondrial function and biogenesis. This study will helpful for analysis of mitochondrial disease.