Exaptation Of Mammalian Wide Interspersed Repeats Biology Essay
The proposed undertaking involves look intoing the function played by Mammalian broad Interspersed Repeats ( MIR ) in human genome. MIR elements are supposed to be originated about 130 million old ages ago and are considered to be the most ancient SINE household.
MIR elements are speculated to play an of import function in development of fresh cistrons by assorted mechanisms. It is speculated that more that 1100 human cistrons have recruited these elements. Besides association of MIR elements with many cistrons associated with morbid status makes it an of import facet to analyze the molecular mechanism behind disease etiology.The rule AIM for the undertaking will beTo measure the preservation of MIR elements in a peculiar cistron in the human genomeTo look into the function of MIR elements in supplying fresh constituents to the endogenous cistrons ensuing into cistron developmentThe above purpose will be achieved by utilizing following experimental techniquesBioinformatics tools like SPIDEY, BLAST, CLUSTALW, REPEATMASKER etcLab techniques like RT-PCR, Cloning, and DNA sequencing ( if clip licenses ) .Background to the ProbeIntroduction:Insistent DNA constitute major constituent of eucaryotic genomes. About 45 % of the human genome is composed of perennial sequences ( International Human Genome Sequencing Consortium ) . These repeated sequences are an of import component in development of complex beings. Many of them are known to play an of import function in development of fresh protein spheres.
Hence the beginning of such insistent sequences and their familial function in assorted facets are of import to understand.Most of the repetition sequences in DNA autumn under two major groups, Tendemly repeated sequences and Interspersed repetitions. Interspersed repetitions are inactive transcripts of permutable elements. It farther includes many subclasses of repetition sequences which are short interspersed nucleotide repetitions ( SINE ) ; long interspersed nucleotide repetitions ( LINE ) , DNA jumping genes and permutable elements with LTR elements. Each category of repetition is associated with certain alone features.
Extensive information on the function played by the repetition component can be obtained if the interpolation of the component is from recent yesteryear as there is less chance of undergoing extended mutant. But to understand the function it is assumed that their function being played is similar to the function it would hold been playing since its beginning or from the clip of exaptation. Older the elements more hard it is to place them and understand their function due to extended alterations in sequence brought about by mutant and sequence alterations by other agencies over the period of clip.Mammalian-wide interspersed repetition ( MIR ) is a category of repetitions belonging to SINEs. In fact MIR is considered to be the transfer RNA derived SINE due to sequence similarity with transfer RNA. It is estimated that MIRs were introduced in the genome of an ascendant of mammals about 130 million old ages ago. It constitutes about 0.4-1 % of the genome of the mammal.
Approximately there are 368000 transcripts of MIR in the human genome. MIR was perchance originated in Mesozoic epoch and was active in ascendants of mammals and birds. It stopped being duplicated before the divergency of placental mammals. Possibly this allowed the elaboration and spread of many specific repetition sequences. ( JOHN R.
MURNANE and JOSE F. MORALES, 1995 )MIR elements were originally observed in a hepatocarcinoma cell infected with Hepatitis B virus and the flanking part of the site where viral atom was integrated were sequenced. In this experiment 70 bp conserved sequence was observed which was non sharing its belongingss with any known category of repetitions. Subsequently this category of repetition was discovered in many human cistrons and on occasion in gnawers. This repetition sequence was found either in noncoding DNA or in the 5 ‘ or 3 ‘ flanking part.
( LAWRENCE A. DONEHOWER, et al. , 1989. )MIR and SINE household of repetitionsSINE are interspersed repetitions of about 100-300 bp which are found in most craniates and invertebrates. One of the omnipresent sequence nowadays in all placental mammals termed MIR shows presence of some of the features of SINEs as concluded by Arian F.A.
Smit ( 1995 ) after alining over 80 sequences incorporating MIR similarities. There was a consensus sequence of 260bp identified and it is expected to be originally transcriptionally active. Besides it has consensus RNA polymerase A and B boxes and an AT-rich 3 ‘ terminal which is a features of typical SINE. Third major feature of direct flanking repetition is likely to hold become unrecognizable since the MRI fragments have really much diverged from the consensus. At the 5 ‘ terminal there was 80bp of MIR that was recognised to be similar to tRNA.
One of the characteristics of MIR transcripts is that they are truncated at either or both terminals but core part was conserved. This could be perchance due to uncomplete integrating procedure. Another ground for this could be that cardinal part is better conserved over terminal sequence as latter shows higher content of mutagenic CpG sites. MIR therefore appears to be a fusion merchandise of transfer RNA derived SINE and unrelated sequence.Distribution of MIR in the human genomeMammalian interspersed repetitions are found in many categories of mammals including placental mammals, pouched mammals and egg-laying mammals. It is considered to be most ancient SINE household. It is believed that elaboration of MIR elements stopped in the ascendants of placental mammals.Human genome is considered to be the mosaic of Deoxyribonucleic acid sections called Isochores.
Distribution of MIR in human genome is explained as per these isochoric parts by Giorgio Matassi ( 1998 ) . It explains the sections of DNA demoing unvarying GC content. On the footing of this human genome is divided into four households of Isochores which are L, H1, H2 and H3 each characterised by different GC content and cistron denseness.Majority of MIR repetitions were discovered in L part of human isochores demoing least GC content and cistron denseness.
MIR denseness was found highest in H2 part which shows relatively higher GC content. MIR elements being discovered in human genome are assumed to be the consequence of retroposition events and farther alterations brought approximately by evolutionary procedure like interpolation or omission of sequences. Important thing to observe that these sequences are still conserved at many places as these are stably incorporate repetitions.MIR elements are found in differential denseness in different isochoric parts.
It is found most often in L isochore and least in H3 isochore. This indicates mobility limitation on MIR elements to travel between different isochoric parts. One of the grounds presumed for this lower distribution of MIR in H3 isochoric part is hapless integrating of MIR in H3 as the size of intergenic part and intronic sequence is smallest in H3 isochore.
Besides intergenic sequences are rich in GC content and 3’untranslated parts which could be involved in regulative function. This could be a possible ground to avoid possible effects of MIR integrating in this part of the genome which might interrupt cistron map.Proposed function of MIR in the cistron developmentThere are many groundss about how a permutable elements by assortment of agencies bring about freshness in protein encoding cistrons by adding new coding DNA to ancient cistrons. Exaptation of Transposable elements, exon duplicate and de novo exonisation from intronic parts can add new coding DNAs to evolutionarily ancient cistrons. Exonisation of archpriest specific Alu component is studied really good in item and a similar procedure of MIR exonisation is being speculated. MIR is now known to play an of import function in look of several mammalian cistrons following one of the mechanisms mentioned below.It may supply alternate splicing sites and hence may bring forth more than one transcript for the same cistron.
It may supply poly A tail for transcripts. It could be involved in presenting new coding DNA in the bing protein coding cistron and hence may present novel spheres in the protein. Besides if it is in the intronic sequences so it may include some of the bases in the transcript and hence may play function in presenting extra protein coding information.One of the observations of MIR indicates possible function of MIR integrating in cistron control and development as proposed by the survey of two cistrons Insulin like Growth Factor 1 ( IGF1 ) and Dendrin cistron. These are good illustrations to exemplify how a MIR component is included in the messenger RNA sequence and therefore became a functional portion of the cistron. ( DAVID C.
HUGHES, 2000 )In IGF1 3 ‘ UTR sequence is about 400 bases long. It shows presence of nucleus MIR sequence of 89 bp conserved in among all species under survey. IGF1 transcript is transcribed and processed into multiple messenger RNA. 3 ‘ UTR of IGF1 transcript is encoded by individual big coding DNA. 3 ‘ UTR consist of many blocks of conserved sequences of about 326 bp which includes MIR. This transcript was found in worlds, sheep and other craniates but non in fish and poulets and hence it helped to pull two decisions. MIR interpolation was early in mammalian development.
MIR integrating resulted in development of new transcript of IGF1.Similar observations made for Dendrin cistron where presence of 2 MIR elements was confirmed by repetition masquerader. Although the map of the peptide coded by cistron is non known but analysis of sequence drew attending to a fresh function of MIR that was predicted.MIR can be inserted in Deoxyribonucleic acid in either sense or antisense strand.
Accordingly it might be included in either sense or antisense orientation in transcripts which can organize heteroduplex. This indicates the importance of conserved sequences excessively. If such heteroduplex offers advantage like in instance of station transcriptional ordinance so flanking sequences may meet to increase sequence specificity and stronger adhering between sense and antisense strand. Although this hypothesis is non so far tested but this can be one of the function played by MIR inserted in this form following mechanism described but extended research is needed in this way if such form of interpolation is observed in some more cistron sequences.
There are illustrations of cistrons where MIR elements were involved in the messenger RNA as a consequence of creative activity of alternate splicing sites. Some of these observations were made in human cistrons as was the instance with acetylcholine receptor cistron. This cistron has gained an extra coding DNA during the class of development due to the new splicing site occurred within conserved nucleus part of consensus sequence.
In add-on as a consequence of curative splicing ~26 bp of MIR component has been incorporated into the coding part of this protein. ( JOHN R. MURNANE and JOSE F.
MORALES, 1995 ) .3 ‘ terminal of MIR sequence is homologous to four complementary DNA sequence. Two among these are human beta tubulin and sheep follitropin receptor. Two others were non good characterised. 3 ‘ terminal of MIR sequence contain poly A signal conserved in all four of these complementary DNA. Hence sequence of MIR component used as poly A signal for these cistrons.
These are the good illustrations to explicate how MIR sequences can play a function in protein development by the presence of sequence resembling poly A tail.There are indicant that exonisation of MIR in worlds is still traveling on. Exonisation of MIR can take topographic point at any point of clip during the class of development and need non be before the divergency of gnawers from Primatess. Like exonisation of MIR in cistron ARNTL occurred before the divergency. On the other manus MIR exonisation was seen in cistron TTLL6 shows presence of splicing site in a testicle specific coding DNA. This functional assignment is observed merely in worlds and hence indicates splicing signal was acquired merely late during human development. ( LAN LIN, et al. , 2009 ) .
This besides indicates how MIR can be a portion of a transcript expressed in tissue specific mode and could hold played an of import function through alternate splice sites.One of the observations made in the MIR component is really unusual in that cardinal nucleus part of MIR is much more conserved over flanking parts of MIR component and possible ground could be those mammalian cells have found usage of these sequences.MIR and DiseasesThere are some indicants that MIR elements are present in some of the cistrons which are of import in morbid conditions. Hence there are guesss about the function of MIR in this facet.
MIR elements are associated with cistrons involved straight or indirectly in morbid status. There are many illustrations for these but some of them are mentioned here.Presenilin2 ( psen2, Website 1, OMIM ID-+600759 ) is a cistron associated with Alzheimer ‘s disease is known to hold MIR component in its cistron. Besides cistrons like ( TGM2, Website 2, OMIM ID- *190196 ) transglutaminase2 is known be associated with many diseases like celiac disease, Huntington disease besides shows presence of MIR component in its cistron. Apart from many other cistrons like Angiotensin change overing enzyme 2 ( ACE2, OMIM ID- *300335 ) , Synaptogyrin1 ( SYNGR1, OMIM ID- *603925 ) besides has MIR in their cistrons.The function of MIR in the disease Chronic Granulomatous disease ( CGD ) is quite clear. In CGD scavenger cells can non bring forth microbicidal merchandises like reactive O metabolites.
This is due to fresh mutant within intron 6 of CYBB cistron that activates deep coding DNA taking to inclusion of this coding DNA in CYBB messenger RNA. Although apart from the pathological status many tissues include this coding DNA in messenger RNA. The part included in the usually expressed CYBB messenger RNA shows individual ORF and therefore it does hold coding possible. This deep coding DNA belongs to part demoing high similarity to MIR. There were no splicing sites or donor sites recognised in the non-primate mammals and Primatess demoing homologues sequences. Besides some interpolations and omissions were observed in these cistrons. Hence it is speculated that some mutants in MIR enabled DNA to be spliced into messenger RNA ensuing into CGD status.
( ANDREAS RUMP, et al. , 2006 )Besides wild type p53 cistron which is a known tumor suppresser cistron is known to demo presence of MIR component at the 3 ‘ UTR part which could be involved in interlingual rendition activation by advancing polyadenylation of messenger RNA. Further probe in this way might uncover some fresh mechanism of p53 cistron ordinance through some novel targeted mutant in this part. ( A. M. D’ERCHIA, et al.
, 1999 )It is assumed that interspersed repetition elements play an of import function in unequal recombination events during meiotic crossing over and which consequences in mutants. As a effect to such recombination event there are happenings of many familial upsets and late shown that it may take to carcinogenesis every bit good. ( Nystrom- Lahti et. Al, 1995 ) . This can be a speculated function of MIR and hence it could be associated with the cause of the disease.Residual motion of MIRIt was proposed that MIR elements stopped being amplified before the divergency of gnawer from Primatess. If so so common ascendant of both gnawers and Primatess must be demoing presence of MIR.
The survey was carried out comparing human and rodent genome.If we propose that heterotaxy stopped after the divergency so none of the species should hold orthologous constituent in the other one but this is non wholly true. If MIR activity stopped before divergency of archpriest and gnawer so there should non be any noticeable similarity between these repetition elements in mouse and human presuming that they were selected neutrally and there was no choice force per unit area. This is because rate of development of gnawers is faster than worlds. Still there was 20 % sequence divergency of intragenomic MIR from their mammalian consensus sequence.
This implies that transcripts of MIR in murine and worlds are under extremely negative choiceBesides at least 44 % of human MIR sequences do non hold orthologue in murine genome. Similarly 16 % of murine genome does non hold any human homologue. This clearly indicates that some of the transcripts were evolved subsequently after the split of gnawers from Primatess. Hence this forces us to suggest that activity of MIR elements have non stopped wholly after the divergency of archpriest and gnawers but it might be demoing some of the residuary motion although this might non be true but it will be clear one time this sort of comparative survey is carried out in other related genomes ( J. C.
SILVA, et al. , 2003 ) .Hence MIR elements are expected to be the category of interspersed repetitions that can lend to the cognition about the exonisation of repetition elements in the human genome. It is non every bit immature as Alu elements where exonisation procedure is non complete besides it is non every bit old as SINEs where extended alterations in nucleotide sequence makes it hard to understand the exonisation procedure. Association of MIR elements with disease doing cistrons makes it an of import facet to analyze about the function played by MIR in development of cistrons.
AIMS of the research undertakingA Mammalian broad Interspersed Repeat is a category of interspersed repetitions that is non extensively investigated DNA sequences to analyze the importance of these elements in the mammalian genome. The functions played by MIR elements in the human genome in the undermentioned facet will be investigated.To measure the preservation of MIR elements in a peculiar cistron in the human genome ( cistron to be decided before get downing the undertaking )To look into the function of MIR elements in supplying fresh constituents to the endogenous cistrons by supplying polyadenylation site, supplying alternate splicing site in order to bring forth population of instead spliced transcripts, or if present in intronic part it can be included in transcript and present excess amino acid and hence can play a function in protein development.
Hence maintaining these positions in front investigate the functional function played by MIR in the human genome if any.To look into the function played by MIR in mRNA localization of function.Experimental attackThe major constituent of the undertaking depends upon the usage of bioinformatics tools and molecular biological science techniques.The first and first portion of the undertaking is the usage of bioinformatics tools to place the presence of MIR in a cistron and besides to derive some information about the function that it might be playing in that cistron. Some of these tools are mentioned here.Repeat Masquerader:It is a plan that screens DNA sequence for interspersed repetitions and low complexness DNA sequence.
The end product of plan is a elaborate note of the repetitions present in the question sequence every bit good as modified version of question sequence where all the repetition sequences are replaced with ‘N ‘ and so the comparings are made between the sequences. ( Website 6 )SPIDEY:Spidey compares mRNA with genomic Deoxyribonucleic acid. It can accomplish two ends while doing this comparing:It can happen alignments regardless of noncoding DNA sizeNearby pseudogenes or perilous can non make jobs in placing coding DNAs and noncoding DNAs. Besides it can execute cross species alliances of messenger RNA to genomic DNA. Hence this tool can be used to analyze MIR sequences among the mammals and between different species. ( SARAH J. WHEELAN, DEANNA M. CHURCH and JAMES M.
OSTELL, 2001ClustalW2:It is a multiple sequence alliance plan for DNA or proteins. It produces biologically meaningful multiple sequence alliances of diverged sequences. It calculates best lucifer for the selected sequence and line them up so that it identifies similarities and differences. Besides evolutionary relationships can be seen by sing cladograms or phylograms. ( Website 7 )Blast:It finds parts of local similarity between sequences. The plan compares nucleotide or protein sequences to sequence database and calculates statistically important lucifers. BLAST can be used to deduce functional and evolutionary relationships between sequences every bit good as aids to place members of cistron households.
There are many types of BLAST but nucleotide BLAST will be used frequently. ( Website 8 )The research lab experiments will do usage of following techniques:Use of Exon Array:Exon array is the look tool to transport out look profiling of a cistron but examining exon instead than merely 3 ‘ terminal analysis as was carried out with traditional micro array surveies. It works makes usage of micro array engineering to accomplish two ends while analyzing genome. One of the ends is to analyze alternate splicing form at the exon degree with more than 4 investigations per coding DNA and allows us to observe specific changes in exon use in a transcript. Second it helps to analyze cistron look as it can observe all the alternate transcripts from a cistron and hence can transport out look analysis more expeditiously mensurating all transcripts from a cistron.
These can be used to analyze inclusion of MIR in a cistron particularly when it is present in the intronic part and portion of MIR is fused with nearby coding DNA and hence it forms portion of mature transcript finally translated into peptide constituent and hence by utilizing this engineering exonisation of Mir can be studied. ( Website -5 )2. RT- PCR:MIR is known to be a portion of transcript if it is spliced instead due to presence of splicing site in the MIR when nowadays in intronic part. Besides from Exon array surveies merely splicing profile of coding DNA can be studied.
It is non possible to obtain information about the inclusion of coding DNA in transcript is constituent or non. It is besides possible that merely in instead spliced transcripts it is included. Second it is possible that MIR may be included in tissue specific mode in a transcript.
Hence to analyze item splicing pattern RT-PCR with complementary DNA sequencing method can be used where it is possible to synthesise complementary DNA from population of messenger RNA of a individual cistron. Besides sometimes it is hard to do usage of this method to analyze MIR coding DNAs near short terminus coding DNA or if MIR coding DNAs with complex alternate splicing events at next coding DNAs. Alternatively cloning of cistron with MIR included in transcript in look vector can besides assist.3.
Deoxyribonucleic acid sequencing:If clip permits so rhythm sequencing or some other advanced sequencing technique can be used to sequence Deoxyribonucleic acid from different mammals to be compared. It can be used to compare orthologous cistrons between different categories of mammals and so function of MIR in development can be studied. This will be needed in instance the genome of the being to be studied is non sequencedMilestones:The Gantt chart explicating the clip program to accomplish the experimental purposes is show below.
Assorted phases of research undertaking:Phase 1: Traning to utilize Bioinformatics toolsPhase 2: To utilize Bioinformatics tools to place cistrons in human genome demoing presence of MIR.Phase 3: Training in experimental techniquesPhase 4: Use of RT-PCR technique to look into the function of MIR on cistron transcriptPhase 5: Deoxyribonucleic acid sequencing if the genome has non been sequenced of the being to be analyzing. ( Not yet finalised, Technique will be performed if clip licenses and besides on the consequences obtained from old phase consequences )Phase 6: Analysis of all the consequences utilizing bioinformatics tools, statistical methods etc.Phase 7: Final undertaking study composing and entry of thesis.