Zebrafish ( Danio rerio ) are the little fish under the group of cyrinide from member of telosts and nowadays in the fresh water beginning from India and Burma. It grows grownups length upto 50mm and life span about 3 old ages. Female fish lays the eggs about 100-200 eggs per hebdomad and can be fertilized externally and develop together. In modern scientific research field, Zebrafish are play an of import function in developmental biological science. Zebrafish embryos are really crystalline and develop really rapidly and it can be observe greatly in developmental procedure and imagination. ( McGonnell and Fowkes, 2006 ) . Zebrafish mitochondrial genome construction consist of 165966p and contains 13 protein coding cistron, 7 fractional monetary units of NADH ( uniquinone oxidoruductase ) , 1 fractional monetary units of cytochrome C ( cytb ) , 3 fractional monetary units of oxidase composite and 2 ATP synthase and ribosomal DNA contains little fractional monetary units ( 12S ) and big fractional monetary units ( big fractional monetary units ( 16S ) and tRNA cistron ( 22 ) . A noncoding part has contain to originate visible radiation and heavy strand reproduction which present inside 5 tRNA cistrons. ( Howley C and Ho RK, 2000 ) .
In female, Oogenesis or egg formation takes topographic point in ovary. Each ovary contain diploid cells called oogonia to compensate from embryologic source cell. Before birth oogonia divided by mitosis. And some of them develop in primary oocyte. Primary oocyte contain immature egg cell contain with monolithic cells called follicle. ( Fleming et al, . ( 1983 ) .Primary oocytes so inner miosis 1 procedure halt uncompleted and their pubescence. No primary oocytes for act in this point. At birth each female has the finite figure of primary oocytes available for reproduction. At pubescence complex signifier to excite alteration in the follicle and give the some primary oocytes complete their mitosis1 activation. The divitional cytol and the cell organell is unequal as consequence one big secondary oocytes and one little polar organic structures formed per primary oocytes. Eppig J ( 2010 ) . The polar organic structure after degenerates the follicle contain secondary oocytes continued material and service to travel the border. Initiate ovulation the mature follicle repture let go ofing the secondary oocytes at this point secondary oocytes start miosis 2 Begins. This miosis 2 will non completed over all unless fertilisation occur. The secondary oocytes envelope the Fallopian tube where fertilisation of the oocytes with sperm cell can happen. The entry of the sperm cell into the cytol of the secondary oocyte strickes the completion of the miosis 2. Cytoplasm divide unevenly bring forth the mature egg cell and secondary polar organic structure. The merger of the monoploid sperm cell and monoploid egg cell has merchandise the diploid fertilized ovum cell. ( Spikings, 2007 ) .
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Structure of an Oovary ( Ova production, 2010 ) .
Ooctyes development in zebrafish consists of 5 phases depends on morphological and biochemical footing. During Primary growing phase ( stage1 ) , oocytes consist of no yolk and looking like a transparent in cytol which is sourrounded by originative cysts. In cortical air sac phase ( stage2 ) , the oocytes are differentiated by visual aspect of different sized cortical air sac and can be seen easy in vitelline envelope. During phase 3 ( vitellogenesis ) , it involves the aggregation of lipoids and yolk organic structures present in the oocytes. Oocytes ripening phase ( present 4 ) Involves the oocytes increase in size and appear translucent. Egg yolks are looking like non-crystalline which admitted meiotic ripening. During mature egg phase ( present 5 ) the eggs are about 0.75mm in size. Howley C and Ho RK ( 2000 ) .
Different phases of oogenesis and egg activation from ( Pelegri F, 2003 ) .
Oocytes are ovulated in the ovarian lms and now it can be capable of fertilisation. The aggregations of maternal merchandises are involved in the procedure of early phase of ovarian follicle development. At this development, the karyon cell are gathered at the boundary of the karyon and it is chiefly affecting the reproduction of ribosomal RNA. After this phase, follicle development are condensed the chromosome and extends the Deoxyribonucleic acid construction which contain proteins and RNA. ( Selman et Al, . ( 1993 ) . For written text and reproduction procedure, RNA and protein are play an polar function in development phase. So isolating messenger RNA from ovarian follicle are really utile in look of cistron in mitochondrial DNA reproduction and written text.
Mitochondria Deoxyribonucleic acid
Mitochondrions are the closed dual membrane and rod-like construction nowadays in the eucaryotic organells. The Mitochondria membrance construction consists of outer membrane and inner-membrane constituents and in between these constituents, inner-membrane infinite is present. The outermembrane is made up of phospholipids and protein chiefly working as the permeable to ions, ATP and ADP molecules. Inner membrane consists of fold like construction called cristae. Cristae are the topographic point or procedure of negatron conveyance concatenation and it generate the ATP production by oxidative phosporylation. This aerophilic respiration procedure are involved in another metamorphosis like anaerobiotic glygolysis and citric acid rhythm and their terminal merchandises are carbon dioxide and H2O. It release 32 molecules of ATP in aerophilic respiration and anerobic respiration generates about 2ATP molecules. ( Oliveira et al, . 2007 ) . By add-on of chondriosomes are included the urea rhythm, fatty acerb oxidization, iron-sulfar bunch, lipid, aminic acid and base and heme biogenesis. Lemires B ( 2005 ) .
Mitochondrions are play a polar function in reactive O spices to actively involved in aging and premature. Mitochondria are the power house of the cell contain molecular machinery for the transition energy from the dislocation of glucose into ATP, the energy currency of the cell. In the two membrane, interior membrane is fold into cristae, provided big infinite to green goodss enzymatic procedure occurs. the interior matrix contain enzymatic system creb ‘s rhythm which contain mitochondria their ain DNA genome and pure indistinguishable molecular dual criterion DNA and all mitochondrial DNA is heritage from female parent and other constituents reproduce by spliting. The biological cell tells the mitochondrial are descented from bacteriums where corporate into the cell in the symbiotic cell. ( Iborra et al, . 2004 ) .
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Structure of Mitochondria from ( Matthew Damstrom Organelles undertaking, 2010 ) .
Mitochondrions are the bantam jammed construction nowadays with the cells that can be change over the energy from nutrient to the cell. Actually DNA is made up of condensed chromosomes inside the karyon and same chondriosome besides have the little sum of the Deoxyribonucleic acid molecules. This familial constituents is called mitochondrial Deoxyribonucleic acid. In the cell, the chondriosomes are present in more than thousand of sum and it is present in DNA round form off from bacterial lineage. ( Mitochondria: construction and Function, 2010 ) .
Mitochondrial cistron are accumulated in the chondriosome chromosome which passes one coevals to the another coevals in most mammals and that heritage in maternal heritage is ground being DNA chondriosome in it. The sperm chondriosome lost when sperm fertilized the eggs. So even though the fertilized ovum bearer the karyon Deoxyribonucleic acid from the female parent cells and indispensable these chondriosomes chromosomes passes one coevals to following coevals it behind to deviate non because cumulative mutant. ( Shourbagy et al, . 2006 ) . Mitochondrial genome which contain dual criterion round Deoxyribonucleic acid about 16.5Kb with differing the size between species to species like human contain 165696bp, the hog contains 16679bp and the mouse contains 162956bp. Nucleic DNA is somewhat different from familial codification to mitochondrial DNA. ( Spikings, 2007 ) . Zebrafish mitochondrial genome construction consist of 165966p and contains 13 protein coding cistron, 7 fractional monetary units of NADH ( uniquinone oxidoruductase ) , 1 fractional monetary units of cytochrome C ( cytb ) , 3 fractional monetary units of oxidase composite and 2 ATP synthase and ribosomal DNA contains little fractional monetary units ( 12S ) and big fractional monetary units ( big fractional monetary units ( 16S ) and tRNA cistron ( 22 ) . A noncoding part has contain to originate visible radiation and heavy strand reproduction which present inside of the 5 transfer RNA cistrons. Howley C and Ho RK ( 2000 ) .
Degree centigrades: UsersMalarvizhiPicturesimagesCA8J4QGR.jpg
The Mitochondrial genome ( Barcoding Life, 2010 )
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Mitochondrial DNA are chiefly responsible for bodily mutant ( non-inheritance ) . Bodily mutant cause the amendss in DNA construction and execute same change and omission in DNA sequence. In regard of this, non passes the functional activity from one coevals to future coevals. Bodily mutant generates the potentially deadly molecules in mitochondrial Deoxyribonucleic acid is called reactive O species ( ROS ) . So it can be easy causes harmful to the mitochondrial DNA. Mitochondrail DNA ( 2009 ) . In chondriosome, more than 1000s of mitochondrial DNA are passed their coevals by motherly completed in cytol of oocytes. If the fertilized ovum can have in add-on of more than one mitochondrial Deoxyribonucleic acid and it will gives the different signifier of cell proliferation and cell division. This causes the different cell line of descents and forms the mutant. So mitochondrial are play an of import function in the cell physiology and bodily mutant or inherited mutant chiefly affecting the developmental abnormalcies. The high grade degree of mutant in mitochondrial DNA molecules causes the amendss during embryogenesis. ( Broughton et Al, . 2001 ) .
Mitochondrial DNA reproduction and Transcription
mtDNA reproduction can happen at any stage of cell rhythm and hence implies that they are independent of the atomic DNA reproduction. In a metabolically active cell bulk of the mtDNA exists as a three-base hit stranded construction called the supplanting cringle ( D-loop ) . The D-loop is a nacent H-strand DNA section of 500-700 National Trust that remains annealed to the L-strand, this constitutes the major non-coding section, incorporating chief regulative elements for mtDNA written text and reproduction. The H-strand encodes most of the information: 14 of the 22 transfer RNA and rRNA-encoding cistrons and the messenger RNA for 12 out of 13 polypeptides. The L-stand encodes staying eight transfer RNAs and one messenger RNA ( Anderson et al. 1981 ) . The cistron merchandises of these messenger RNAs are indispensable for the map of the chondriosome. The major difference between the mtDNA and the atomic Deoxyribonucleic acid is the compact cistron organisation where in all the cryptography sequences are immediate to each other separated by a few bases and without noncoding DNAs ( Zeviani et al. 1998 ) .
There are merely two non-coding parts in the mtDNA which play an of import function in DNA reproduction. One is the D-loop part situated between cistrons that encode transfer RNA for phenylalanine and proline. This part contains the beginning of reproduction for the H-strand ( OH ) and the boosters for the H- and L-stand written text named PH and PL severally. The 2nd non-coding part is a much shorter section of DNA which is approximately 30 bases long. This part contains the beginning of reproduction for the L-strand ( OL ) . This part is located inside a tRNA bunch 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 denseness in denaturing gradient giving rise to Heavy or H-strand and light or L-strand.
The written text from both the PH and PL boosters proceed through the full mtDNA and consequences in a polycistronic RNA. The transcripts contain tRNA cistrons between the larger rRNA and messenger RNA sequences. The folding of tRNA AIDSs in the cleaving of the messenger RNA and rRNA from its polycistronic transcript. These messenger RNAs are subsequently post-transcriptionally modified by polyadenylation and the transfer RNA by add-on of 3 ‘ terminal CCA ( Taanman 1999 ) .
Transcription and reproduction of mitochondrial DNA plants under the nucleus-encoded protein inside the part of the D-loop supplanting. In the procedure of written text required the elements like Mitochondrial RNA polymerase, TFAM Aand TMB1M or TMB2M.Replication of Mitochondrial DNA is chiefly involved in nuclear-encoded protein, particularly mitochondrial DNA specific polymerase gamma. Polymerase gamma are consists of two fractional monetary units, they are POLGA and POLGB. POLGA are catalytic subnit which is subjected to elaongation of new girl bases and POLGB are accessary fractional monetary units which is involved to primer individuality and cogent evidence reading procedure. . The primer for induction of DNA reproduction involved by process of light strand transcripts with the Mitochondrial RNA Processing. Thus the demand of the transcript to bring forth primer high spots the inter-relationship between written text and the mtDNA reproduction.
Mitochondrial DNA Replication and Transcription taken from the image ( Spikings, 2007 )
Mitochondrial DNA reproduction procedure has two mechanism. First one is asynchronous displacement replicaion mechanism and 2nd one is taking and lagging strand mechanism.The reproduction of mtDNA is initiated at OH utilizing the above mentioned primer and returns displacing the parental H strand. The expansion of this supplanting cringle ( D-loop ) towards the OL part is two tierce of the length of the mtDNA. The supplanting of the H-strand exposes the OL part which forms a root cringle construction taking to the induction of L-strand synthesis which proceeds back along the open individual stranded H-strand templet 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 reproduction.
Recently another mechanism of mtDNA reproduction has been proposed called the leading and lagging strand mechanism by Holt et Al ( 2000 ) . In this theoretical account, both the strands of mtDNA is said to be synthesized bidirectionally and the two-base hit stranded reproduction fork returns through the length of the mtDNA. Here zone of reproduction is believed to be present beyond the D-loop. Exp-D and GpC signifiers are excluded by this manner of reproduction. ( Spikings, 2007 )
Polymerase Gamma ( PoL I» )
Mitochondria DNA is polymerized by mitochondrial specific DNA polymerase gamma, ( DNApoli»» or PolG ) and belongs to the heterotrimeric enzyme. The enzyme consists of two fractional monetary units, the big catalytic I± fractional monetary unit of 125-140 kDa and the smaller I? fractional monetary unit of 30-54 kDa. The I± fractional monetary unit ( catalytic fractional monetary unit ) is responsible for elongation of the girl strands where as the I? fractional monetary unit is indispensable for cogent evidence reading and primer acknowledgment. I? fractional monetary unit ( accessary units ) is called the fix enzyme which actively involved repair mechanism of the polymerase. ( Oliveira et al, .2007 ) . In chondriosome DNA, DNA polymerase I? are chiefly started the elongation procedure in part of D-displacement loop.Apart from the DNA polymerase reproduction is besides dependent on other proteins like mitochondrial helicase. mitochondrial individual stranded adhering protein and DNA primase involved. ( Spikings, 2007 ) . Deoxyribonucleic acid polymerase gamma are place of topographic point for the mitochondrial disease around 100 disease occurred by mutant and disease like Neuropathy, syndrome, Alpers syndrome, midlife-onset ataxy and male sterility. so we can easy calculate out the mechanism of mutant by which cistrons caused the instability of mitochondrial DNA. So DNA polymersase I? are play an of import function in reproduction of chondriosome DNA and If any alterations or mutant occur in the catalytic fractional monetary units will do mitochondrial upset. So we can analyze these cistrons with extended observing techniques like RT-PCR, Microarray and Northern blotting. Chan and Copeland,2009 )
TFAM
TFAM ( Mitochondrail written text factor A ) are type of protein belongs to HMG ( high mobility group ) membrane and is chiefly involved the mitochondri DNA maintainance. It has 2 HMG box-domain. HMG protein are respondible for wrapping, wind offing DNA sequence and binding. ( Alam et al, . 2003 ) In mitochondrial DNA, TFAM is play an polar function in mechanism of reproduction and written text. When TFAM degrees are increased within chondriosome, it will increase the degree of mitochondrial DNA reproduction and mitochondrial DNA written text. It is really of import for embryologic development. When TFAM degrees are lessening, it induces the assortment of mitochondrial upsets like familial mitochondrial upsets like familial mtDNA liver disease, childish mitochondrial myopathy, skeletal musculus, fatal childhood myopathy, mitochondrial encephalomyopathy disease, musculus cachexia and optic myopathy. And besides cut downing the TFAM look in chondriosome when spermatogenesis in mammals. This will cut down the mtDNA transcript figure and diminish the transmittal of paternal mtDNA to one coevals to future coevals. ( Spikings, 2007 ) .
House maintaining Gene
House maintaining cistron are the type of cistron which is present in all tissue and keep the cellular map. These cistrons are merely present in the peculiar organ or tissues and affecting the peculiar development map and cellular activity. Gene look is really of import for analysis normal and unnatural biological status. Comparing between housekeeping cistron and peculiar tissue cistron are really indispensable. So these cistrons are play an of import function to analysis the normal functional status of peculiar tissues. For look of cistron, there are so may techniques used for biological research, clinical usage and diagnosing chief techniques like RT- PCR, Northern blotting, microarray and western blotting. The most familiar house maintaining cistrons are I?-actin, I?-tubulin, GAPDH and EF1I± ( Ferrari, 2005 ) . Actin is the ball-shaped protein present largely in the eucaryotic cell. Actin has two types of fibrils viz. cytoskeleton and microfilaments present in the musculus cell. EF1I± is the cistron that responsible for elongation of aminoacyt transfer RNA to the palce of ribosome. Isoform ( alpha 1 ) are present in placenta, kindney and liver. Isoform ( alpha 2 ) are presented in bosom and encephalon. EFA1 I± are largely presented multiple transcripts in chromosomes. ( NCBI,2010 ) These two housework cistrons are play an of import function in mitochondrial DNA reproduction and written text.
Real Time PCR
Real clip PCR is the modern technoique and extremely flexible to investicate, magnify, observe, manipulate and analyses from different beginning. Real clip PCR are fluorescence technique labeling the dual criterion DNA sequence by utilizing DNA intercalating dye and Sybr green. Traditional PCR are observing the amplifier Deoxyribonucleic acid at the terminal of the reaction. It is non giving the proper consequence. But existent clip PCR are really sensitive and observe the big measure of amplicon at the clip of reaction. ( Newton and Graham, 1994 ) . Real timePCR is the quantitative technique to mensurate the sum of messenger RNA or complementary Deoxyribonucleic acid in the given sample from tissue or individual usage. Real clip PCR are chiefly used to mensurate the cistron look of messenger RNA at certain status. Real-time PCR, ( 2008 ) .
For this survey, we are utilizing Zebra fish and will pull out the ovarian follicle at different phases of development and measure their viability and analysis with fluorescent spectrometry. From this we will pull out the RNA and it will change over onto complementary DNA by rearward written text. After that we will utilize existent clip PCR and will show the TFAM, POLGA and POLGB cistrons.
Methodology of this proposal is
Collection of ovarian follicle from zebra fish
Check the viability of follicle utilizing Trypan Blue viabible trial
Extract RNA
Convert RNA to cDNA utilizing rearward written text
Apply in existent clip PCR
Expression of TFAM, PolGA and PolGB
Aim and Hypothesis
The Proposal purpose is to find the mitochondrial DNA factor and to mensurate the look of TFAM, POLGA and POLGB and comparison with housekeeping cistron I?-actin and EF1 I± by utilizing the theoretical account of Zebrafish ovarian follicle and with the aid of the existent clip PCR. From this survey we will analyze mitochondrial map and biosynthesis. This survey will helpful for analysis of mitochondrial disease.
