1. Promoters show the transcriptional initiation site of a gene and are essential for its expression. They have binding sites for basal transcription factors and for RNA polymerase that will bind around the start point. However, in multicellular eukaryotes, many promoters are not sufficient and not capable to drive high level of transcription of the gene. Therefore, their activity is regulated and increased by enhancer sequences. Enhancers can be located within the gene or long away from the promoter, at either site or in other chromosome. In many cases, enhancers fall within introns as well.
Enhancers share common sequences with promoters, but these sequences are more closely arranged. They hold binding sites for transcription factors which will in turn bind on TATA box (in the promoter of the gene) and drive expression. So by binding to enhancers, transcription factors (activators and coactivators-like TFIID) can easier bind to TATA box and interact with the RNA polymerase II and the other factors that are on the promoter. Enhancers increase the amount of activators and antagonise silencers that can interfere with them. Repressors bound to silencers can lead to the opposite effects than an enhancer. They decrease the transcription of a particular gene. Enhancers prevent repression of transcription that results from the condensed structure of the chromatin.
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Enhancers have tissue specific activity and temporal regulation. This means that they boost the transcription of a gene only if they are present in specific tissues and when the gene product is necessary. This is due to the common sequence elements that enhancer and promoter share. These are the transcription factors (activators) that are activated in certain time and tissue. They bind to enhancer and promoter and via coactivators, all the proteins interact and regulators can then bind and change the chromatin structure to facilitate transcription. In this way, the transcription pattern of gene regulation can be followed in both space and time.
An example of specific transcription is the one for the immunoglobulin genes. These genes are only active in B-lymphocytes and not in other cells because their expression is controlled and activated by both enhancer and promoter present in the transcription unit. If the same enhancer is taken and located to other cell line, then the immunoglobulin genes will not be transcribed. Some other examples of enhancers in genes that are expressed in certain tissues is the a-1-antitrypsin in the liver and insulin from pancreas.
2. A promoter is required to be adjacent to the coding sequence of a gene in order to drive transcription. An enhancer on the other hand, can activate a promoter even if it is many kilobases away from it. In the case of the enhancer trapping vector, the P-element holds the promoter and the enhancer will be detected even if these two elements are not very close together. Enhancers can be upstream or downstream of a promoter, in either orientation. Even if enhancer is several kb away, it can come close together and act on the promoter. Enhancer provides binding sites for transcription factors. Many proteins will bind together and will form the enhanceosome. Promoter will also bind proteins that have an affinity with the enhanceosome. The interfering DNA is looped out and the distance sequences are brought close to the promoter. In order to have transcription, the RNA polymerase and basal factors will bind at the startpoint of transcription and at the TATA box. Activators on the enhancer will bind to coactivators and bridge between the activators on enhancer and the basal factors will be formed. The proteins interact because the DNA between them is looped out. The enhanceosome will also recruit the histone acetyltrase (HAT) that will open up the chromatin to aid the RNA polymerase II to act on the naked DNA and initiate transcription.
3. P-elements are inserted at random positions within the genomic DNA. This means that they might land in the transcription unit of a gene and disrupt it, or within a non-coding sequence. If they fall into a non-coding region where these sequences have no function and no product, this will probably have no effect on the phenotype of the animal. On the other hand, if a gene is disrupted due to the insertion event, its transcription and translation will be affected. The amount of protein produced will also change and by not having the correct amount of protein in the pathways that are used, then altered phenotypes, diseases or lethality may arise. Moreover, the structure of the protein might also be altered.
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If the transposable element is inserted into a promoter region, then the transcription of the gene will be either reduced or entirely lost. Therefore, the protein will be either produced in very low concentrations or will not be produced at all (loss of function mutation).
If the P-element is landed within the coding sequence of a gene (exon), this will lead to the production of a truncated protein or to the production of a fusion protein. This protein will contain both the endogenous protein transcribed from the endogenous gene and the protein produced from the reporter gene. This insertion event will cause the loss of function of the gene.
The transposable element can also be inserted into an intron. This will affect the splicing of the protein because the following exons might not be transcribed. A truncated protein or a product that cannot function at all may result.
What is more, an mRNA without a 5'-cap might give rise to a truncated protein because of the fact that the P-element fell in the 5' untranslated region of the gene.
Last but not least, the transposable element might be inserted in a region between the enhancer and the promoter, making more difficult their interaction. This will not allow the enhancer to increase the transcription levels of a particular gene, leading to once again to the incorrect amount of the protein produced.
All these insertion events can be mutagenic, since they alter the production of important proteins that are needed to be present at specific times and certain tissues in the organism.
4. P elements might disrupt the function of a normal gene, depending on the place they land within the genome. They will usually cause a recessive mutant phenotype. If there is a dominant homozygote genotype (wild type or mutant) or a homozygote recessive mutation in the genome of the organism -which does not yet contain the P-element-, the resulting phenotype from those genes will be visible in the animal. If now a P-element is inserted and a new mutagenic phenotype is observed, this will probably lead to the conclusion that the new phenotype that is now visible has arisen due to the P-element insertion.
When a recessive mutant version of the gene already existed in the genome, its effect was not visible because the dominant wild type version of the gene was present on the other chromosome (i.e: heterozygote Aa). When the P-element lands and disrupts the wild type gene, then the function of the wild type gene is lost. The mutant recessive gene that was already on the other chromosome but was masked from the normal allele will now be allowed to show off its action and will produce a recessive mutant phenotype. Therefore, a mutant phenotype to occur, both of the alleles must be mutated.
5. Transgenic constructs can be prepared and popped into the genome of the fly in order to trap the pattern of an enhancer's activity. The transposable P-element has a selectable marker (white gene) in order to identify those flies that have taken up the transgene, and the LacZ reporter gene. This reporter gene is bound to a minimal promoter that on its own is not capable of driving transcription of LacZ gene at a reasonable rate. If now the enhancer trap element is landed within the influence of an endogenous enhancer, the activity of the promoter will be upregulated and beta-galactosidase will be produced. This will be visualised with the use of X-gal that will form blue cells. So, without knowing where and what the enhancer does, its activity is now trapped and can be followed through development of the animal. This means that with this method the expression of the genes is reported; and so the genes' identity. The sequence that is inserted tags the gene.
What is more, P elements are preferred to be used rather than chemical mutagens, because although their mutation efficiency is lower, the genomic sequences adjacent to the inverted repeats of the enhancer trapping element can be simply sequenced with the use of plasmid rescue or inverse-PCR. Moreover, because transposase can mobilise the element, if an element that gave rise to a mutant phenotype is now excised and the phenotype is restored, then it is clear that the resulted phenotype was due to the specific insertion of the transposable element. If the excision of the P-element is imprecise, then the genomic sequence will be altered and maybe a new phenotype will arise which will be of worth to analyse. P-element insertions can also be mapped and find the location on the chromosome that they have landed.
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In other words, screening for mutant phenotypes that have been arisen due to transposable elements, is much easier and safer than for those that chemical mutagens give rise. You can disrupt just one region every time and deduce which gene is affected. Chemical mutagens are toxic. They destroy the DNA at many sites; therefore the reason for the mutant phenotype observed cannot be easily deduced.
6. Mobile genetic elements can give rise to mutations and chromosomal rearrangements. Natural P-elements are appropriately spliced in the germline in a way to produce the enzyme trasposase and allow jumping events to take place. If natural P elements are already in Drosophila, then the artificial inserted P-element will not be stabilized in a place. Our P-element cannot produce this enzyme, so it cannot change position if the P[D2-3] is not present. If many natural P-elements are present, then all of them will jump in the genome producing many new phenotypes. Therefore, it would be impossible to identify the flies that carry new insertion due to the P-element of interest and we would not be able to find the insertion point of this specific element. Therefore, it is required to have only the P-element of our interest in order to avoid the jumping events of many transposable elements. The resulting mutation will be due to the insertion of the artificial P-element and the phenotype will be stabilised.
7. The reporter gene on the transposable element has a minimal promoter that cannot induce on its own at a sufficient level the transcription of the LacZ gene. If an endogenous enhancer is trapped, then it will act on the promoter and increase the transcription level.
To modify the enhancer trap element so that it would act as a promoter trap element, the minimal promoter should be removed. So, except from the marker gene, a promoterless reporter gene should be included in the transposable element. In this way, the only way that the reporter gene is going to be transcribed is to land downstream of an endogenous promoter. Promoters do not act like enhancers which can influence the promoter from a distance. A promoter can initiate transcription of the gene only if it is located adjacent and upstream of the coding region of the gene. This means, that the P-element must fall downstrean of the promoter of a gene X within the coding sequence (in an exon). A fusion protein will then be produced, because both lacZ and gene X were transcribed from the same promoter.
8. The chance that the promoter enhancer element will land within an exon downstream of a promoter is very low, since most of the DNA is consisted of non-coding sequences. This element must be located at that certain region in order to have the transcription of the reporter gene. At the contrary, an enhancer trapping element does not need to fall next to the enhancer; it can be landed either upstream or downstrem, near or far away since the looping formation of the DNA will allow their interaction. So a chance to trap an enhancer instead of a promoter is much higher.
9. Females carrying four copies of the P[lac w+] in each of the X chromosomes were mated with males that had stubble bristles and contained in their third chromosome a transposase. The source of the enzyme is a P-element that differs from the wild type P-element. The transposase is not expressed only in the germ line but also in somatic cells. The male progeny of this cross had stubble bristles and the source of transposase (P[?2-3]) on the third chromosome. Stubble allele is carried on the third chromosome, as well. They also carried the P[lac w+] element on their X chromosome, transmitted from their mother. Because these flies have the transposase, jumping events will take place both in somatic and germ cells, in autosomes or sex-chromosomes. Hybrid dysgenesis occurs. To discover the new transposition events these males were crossed with homozygote virgins for the eye colour mutation w1118. The male progeny of this second generation did not have stubble bristles and so neither the transposase source. Interestingly, they carried the P-element with the wild type activity of the white gene on an autosome, indicating that this element is now in a new insertion site.
If the P-element was not excised from its original region, then it would have remained on the X chromosome. If that was the case, then the male progeny Sb+ from the second cross would not show w+ activity, since their X chromosome was derived from their mother (homozygote for w1118 ). The P[lac w+ ] is the only source for having the wild type activity of the gene. So, by crossing the jump-starters of F1 with the homozygote females for the w gene, the new transposition sites of the P-element carrying the w+ were recovered.
10. In cross1, homozygote females carried four copies of P[lac w+ ] element on each X chromosome. One X was inherited to the male progeny (w+Sb). These males will have all four copies of the P-element if during female meiosis crossover events did not change the position of any P-element. If unequal crossing over occurred, then maybe some P-elements from one chromosome changed position and landed to the homologue. Moreover, these males carry the transposase, hybrid dysgenesis will occur after mating them with the homozygote w1118 females and the P-elements will mobilise to new sites on any chromosome. The transposase that is present is active not only in the germline, but also in the somatic cells. The male progeny from this cross, will have new random insertions of the P-elements, resulting in different eye-colour phenotype. Some of them will have pale yellow eyes, others pale orange and others dark orange. The P-elements contain the functional copy of the white gene. This wild type of the white gene produces the red colour of eyes. The colour that is going to be observed on flies can vary from white to red, including intermediate colours like yellow and orange. This depends upon the location of the P-element and the allele of white gene present on the X chromosome, which in this case is w1118. Hemizygotes for this locus have white eye-colour. But because the P-elements with the functional copy of the gene are present, then the phenotype will not be white. The location where the transposable elements fall will affect their transcription levels. If they fall into regions near heterochromatin, then they will be less transcribed and the product will be reduced compared to those P-elements that landed in regions adjacent to euchromatin. These elements, will be highly transcribed and expressed. Therefore, the more the P-element is transcribed the more protein will be produced. The resulting fly will have darker eyes. This is known as the dosage effect.
Furthermore, the number of the P-elements present in each fly will play a role on how dark the pigment of the eye will be. If more P-elements are present (i.e all four copies) then the chance to fall in sequences that are actively transcribed is greater that the chance that three or less P-elements will have. Again, the less product produced from the functional white gene, the lighter the colour of Drosophila's eye will be.
11. The male progeny of cross 2 carry the P-element on an autosome but not the transposase. This means that the transposable element is stabilised on its new position. These individuals show different eye-colour indicating different insertion events. However it would be possible to derive two or more males that carry the same insertion event. This indicates that at least, two sperms carried the same insertion event. So, somewhere during spermatogenesis, two P-elements were present in homologous loci, one in each homologue and because the transposase was not present in that cell, the P-elements could not excise. This suggests that during spermatogenesis, transposase can be removed.
The diploid spermatogonium during spermatogenesis undergoes a mitotic division and the diploid primary spermatocytes will be produced. Every single primary spermatocyte will go through first meiotic division to produce the secondary spermatocytes. These will now undergo second meiotic division and the spermatids will be produced that will give the sperm cells.
All males w+Sb carry the transposase. This will allow the mobilisation of the P-elements. The transposase is present in the spermatogonium and in the primary spermatocyte which resulted after a mitotic division. Since the P-element on the third chromosome that carries the transposase cannot excise its self, then after mitosis, both the primary spermatocytes will carry the transposase. This means that P-elements can mobilise and land in new positions. During the first meiotic division, the P-elements can fall opposite on the sister chromatids, disrupting the same sequence of DNA. After meiosis 1, the haploid secondary spermatocytes will each half of the chromosomes. This means that transposase that is present only on the one third chromosome (not in the other homologue) will be transmitted and present only to the one secondary spermatocyte. The one that will not contain the transposase and has also P-elements on the sister chromatids that now can not be mobilise, will produce two spermatids (after meiosis II ) tha will contain the same insertion.
This means, that only two flies will carry the same insertion. More than two will be produced, if by chance, a P-element falls in the same position in another cycle of spermatogenesis.
12. The red eye colour in Drosophila, is due to the effect of two different pigments which are produced from different pathways, the ommochrome and the pteridine pathway. These at the end come across and cooperate in the same transport system. This mechanism takes partially place on the X chromosome. The pathway that ommochrome is involved gives the brown
(ommochrome) pigment and pteridine pathway produces the drosopterin-scarlet pigment (bright red) after producing the blue and yellow pigments. These brown and scarlet pigments will then transport to the crystals of the cells in the eye tissue and produce the red colour.
When the P-elements were mobilised and reinserted in new locations, they have probably disrupted genes that were producing enzymes necessary for the two pathways.
When the gene disrupted is one that produces enzyme necessary for the transporter system then the pigments cannot travel to crystals and the colour of the eye will remain white. If the genes that are disrupted are for the production ofenzymes in ommochrome pathway and indrosopterin pathway, then the two pigments will no be produced and again the eye-colour will be white.
The w+Sb males had patchy eyes. A part of their eye was white and some other region was red. Since P[?2-3] transposase was present, then mobilisation of the P-elements was induced not only in the germline but also in the somatic cells. This suggests that during the development of the eye, transposition events were taking place, giving rise to two populations of cells. Those that carried the P-element and could give the wild type phenotype and those cells that lost the P-elements and gave the white colour. Therefore, mosaic eyes were formed. The fact that the P[lac w+ ] element was excised during the development of the animal, gave w white cells on a background of cells that were red. The eye will show less red pigments if the P[lac w+ ] element is excised very early during the development of the eye.
13. The PlacW element was inserted into the genome of D.melanogaster and we were interested in identifying the region where the transgenic insert landed. Therefore, it is necessary to find the sequence of the genomic DNA that is adjacent to the inverted repeats of the P-element, because these sequences before the insertion were next to each other. The sequence of the P-element is known. We digest the genomic DNA with restriction enzyme that is already known that it digests the P-element in known region. From all those fragments that will be produced, only two that are unique are of our interest. These contain the junction of genomic DNA and the 5' or the3' end of the P-element. Ligation of the two ends of the fragments is then followed to produce circles and with the use of specific PCR primers either for the fragment with the 5'end of the P-element or for the 3'end, the fragment is amplified. The circles helped in order to have the primers pointing to the known piece of sequence (the transposon).In other words, they must point outwards the P-element.
In this way, what is amplified is the genomic region. Cycle sequencing is followed to identify the sequence of the genomic DNA. This time, new primers are used (sequencing primers) that are near the original primers. These anneal closer to the genomic sequence, in order not to amplify much of the transposable element and have errors during the sequencing procedure. It is important to sequence the genomic DNA and a less DNA amount of the p-element, so to be more specific in the BLAST search.
It is required to use primers that point outwards the P-element in order to amplify the genomic DNA and sequencing primers that are as close as possible to the genomic sequence, either close to the inverse terminal repeat of the P-element, or close to the restriction site where the enzyme digested the P-element. After ligation and PCR reaction, P-element sequences are at the edge of the molecule, while the genomic DNA is in the middle. Plac1 primer can not be used as a sequencing primer,because although it points to the genomic DNA, it is not very close to it. In this way the DNA sequencing will not be specific for the genomic DNA.
In some other case, using different primers, if the restriction enzyme digested within the P element very close to the inverted repeats, then the same primers can be used for both PCR and sequencing, because what ligated circles will include is a very small fragment of the P-element and a long sequence of the genomic DNA that we wish to identify.
SECTION 2: RESULTS
PlacW is the transgenic construct that is inserted into Drosophila genome in order to trap enhancer sequences. It has inverted terminal repeats and contains the functional white gene that confers to the fly red-coloured eyes. It also contains a reporter gene (lacZ) linked to a minimal promoter, which will drive gene's expression only when the P element is captured within the activity of an enhancer. The pattern of the enhancer activity can be observed and followed by staining the animals with X-gal, at different developmental stages.
The small P-element of known sequence is landed in the huge genomic DNA sequence in a position that is not known. It is of our interest to find out the site of insertion and this is achieved relying on the formation of circles with the use of inverse PCR reaction.
Genomic DNA was extracted from heads of four different Drosophila strains (59, 60, 70 and 83) that contained the PlacW enhancer trap element. Restriction enzyme digestions with two different enzymes HinP1I and Sau3AI were performed in order to generate a fragment that contains the junction between one end of the element and the flanking genomic DNA. This fragment will act afterwards as a template for PCR amplification.
Following digestions, ligations were performed for samples 59 and 60 in order to convert the linear fragments into circular molecules needed for the inverse PCR reactions. It is important to ligate the two ends of the same fragment. For inverse PCR, two sets of PCRprimers were used, specific to anneal either to the 5'end of the P-element (Plac4+Plac1 that work at 60°C) or the 3'end (Pry4+Plw3-1that work at 55°C). In this way the only circles that are going to be amplified are those that contain the inverted terminal repeat of the P-element and the flanking genomic DNA. It is essential that the primers point towards the ends of the P-element, ensuring that what is going to be amplified is the genomic DNA.
After capturing the piece of DNA of our interest, DNA sequencing is performed with primers that will anneal on the P-element, as closer to the genomic sequence pointing to it.
Because DNA sequences from samples 59 and 60 failed to be sequenced, a backup sequence (sequence 1) is provided to perform BLAST analysis. This sequence is shown below and was yield with sequencing primer Splac 2. It is known that the resulted fragment was produced after digesting the genomic DNA with Sau3A.
P-element and genomic DNA were digested with Sau3A, therefore sticky ends were produced. After ligation and after inverse PCR, what is captured is the genomic DNA which is bound to the 5' terminal inverted repeat of the P-element. When the circles were produced, the 5'sticky end of the genomic DNA ligated to the sticky end of the 3'end of the P- element.
The genomic DNA fragment is 173bp long. The total fragment that was sequenced was 259bp long, but the first 86bp correspond to the P-element terminal repeat.
The genomic DNA has a Sau3A restriction site, after the first 41bp. This was yield when ligations were taking place. Many fragments after digestion with Sau3A were produced with sticky ends. What was important was to ligate the two ends of the same fragment. In this case, the fragment near the sequenced genomic DNA that flanks the 5'terminal inverted repeat of the P-element ligated to the sequenced genomic DNA. Therefore, the circle contained the inverted repeat of the transgene and two pieces of genomic DNA.
Using both of the genomic sequences in Blast, the same sequence was found. Therefore, all the 173bp were used in BLAST for further analysis.
1. The gene found using BLAST is referred with the symbol D.mel CG15382 and its sequence location is on the long arm of the second chromosome at 2155760-2156791. It is a protein sequence gene, however its function is still unknown. It has the cytological map location 2L:22D1. This means that the insertion has been positioned to the left of the second chromosome, 22 cM away from the centromere (in the D1 of the 22nd band).
Cytological map shows where the genes are localised on the chromosomes. Deletions, translocations or insertions can be mapped, using G-banding technique that produces a banding pattern. This method is used to stain metaphase chromosomes. DNA regions that are gene rich appear less dark than other regions.
Polytene chromosomes are huge chromosomes that have undergone multiple rounds ofreplication, without cell division. These are the chromosomes that are used to identify chromosomal rearrangements, since they have many light and dark banding patterns. To create the cytological maps, the size of each polytene band is estimated (in Kilobases).
2. There are not any transposon insertions in the area.
There are two genes close to D.mel CG15382 insertion. The gene known as AIF is an Apoptosis Inducing Factor that promotes programmed cell death under certain stimuli and it is found on chromosome 2L(2151668-2155390) (plus orientation). AIF is a flavoprotein that under normal conditions, remains in the intermembrane space of mitochondria. However, when the cells are under stress, it acts as a proapoptotic factor, it translocates both to cytosol and the nucleus and promotes caspase-independent pathways for peripheral chromatin condensation and DNA fragmentation. What is more, it has an effect on the mitochondrial membrane which can be permeabilised and also shows NADH oxidase activity.
The second gene is the aop -anterior open gene, which is in closer vicinity with the insertion CG15382 (2L: 2156484-2178749/ minus orientation). Aop belongs to the family of ETS transcriptional repressors. This means they have specific DNA binding sites that inhibit the transcription and therefore the expression of genes. It plays a key role in the decision that cells have to make; to differentiate or to keep on dividing and proliferating. When it is inactive, differentiation is allowed to carry on. When it is active, it inhibits neural expression and other developmental processes. This transcription factor is needed in order border cells to migrate during developmetnoogenesis and has different expression levels as the cells migrate from the anterior to theposterior site of egg. An example is the effect that aop has on posterior Engrailed cells. It should not be activated in order to allow cells to adopt their correct fate. Aop is negatively regulated by the cytoplasmic pathway Sevenless/Ras/MAPK.
- AIF and aop have been previously studied extensively, since they have important biological functions. Their molecular processes and functions have been publicated. Aop is also known as yan.
- AIF gene is biallelic and produces two proteins, AIF-PA and AIF-PB that are 674aa and 739aa long respectively. Aop gene produces two products, Aop-PA and Aop-PB of 732aa each. In both cases, both products produced from the same gene have the same function.
Apoptosis-inducing factor AIF has three domain. An oxidoreductase domain at the carboxyl-terminal end which when it is mutated, AIF is not capable for death promoting activity, a mitochondrial localization signal (MLS) which is a peptide directing to the mitochondrion and also a nuclear localization signal (NLS) (27 amino-acids long) which is a peptide directing to the nucleus. The translocation depends on the stimuli. Under normal conditions, the protein is in the intermembrane space of the organelle, while in cancer cells it translocates to the nucleus to affect the DNA.
Anterior-open gene (aop) has two domains, a SAM/Pointed domain and an ETS domain ETS domain allows the protein to bind to DNA, therefore aop is a transcription factor. The protein in this way represses the expression of genes. SAM (sterile alpha motif) is involved in protein/protein interactions.
- Both genes (AIF and aop) match to plenty of ESTs. Approximately 15 and 80 ESTs are aligned to AIF and aop gene respectively. Table 1. Shows someof the ESTs aligned to the two genes.
- Expressed sequence taqs (ESTs) are partial cDNA sequences that point directly to an expressed gene and when translated, many together provide many and useful information about proteins. Using EST libraries, the structure of the transcripts can be established and accurately analysed. Most of their sequence is free of repetitive DNA and represent unique short regions of DNA. ESTs are short to make sure that the ends of the sequence are contiguous in the genome. This means that introns do not separate the ends. A large gene can be characterised by many ESTs, that might correspond to many parts of the transcript or transcripts that arose due to differential splicing.
Since many ESTs collections represent many genes that give rise to functional proteins, many phenotypes and physiological responses of an organism can be analysed. They are also useful for the observation of phylogenetic relationships and evolutionary processes, since they are used as genetic markers.
BLAST search does not provide informations about when and where the apoptotic inducing dactor (AIF) is expressed. However, ti is known that AIF in normal cells, is present in mitochondria. In cancer cells it is released from the injured organelle to cytosol and nucleus.. It acts both as a proapoptotic factor and as an endonuclease, by acting on the DNA and induce condensation of chromatin and degradation of the genome, resulting in regulated cell death.. Embryonic stem cells that do not express AIF are resistant to apoptosis.
Aop gene is expressed during the pupal stage in pigment and cone cells, during embryonic stage 10 in the epidermis and in stage 13 in the tracheal system. They are also expressed during larva stage in the eye and antennal disc. The time of expression of this gene is related to the protein's function. Aop during the early stages of the eye development, is active in order to inhibit cell differentiation. Snce it has an ETS binding domain, it can repress the expression of essential genes. Aop (or yan) It is a negative regulator of R7 cells and cone cells, therefore photoreceptor development is repressedr repressors, Ras/MAPK pathway lead to the phosphorylation of aop, and therefore its inactivation. During normal development, when the protein is dephosphorylated (this means the Ras/MAPK is off), aop is active and inhibits both cell division (proliferation) and differentiation.