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Now, we will focus on the reverse genetics based approaches.So, in reverse genetics based approaches here we have first we identify the gene and thenwe try to understand what is the specific function a particular gene or a group of geneshave in in a particular developmental processes.So, if you look this slide, so this process of reverse genetics can be studied in followingsteps.In first step what we aim?We aim to identify a gene of interest and isolate that gene of state; gene of interestfor studying about its function.Then second step of this is that to analyze the expression pattern of this gene.So, here we are starting from a gene.So, we should know where exactly this gene is expressed particularly with respect tothe developmental stages, developmental tissues organs whatever it is.Then once we identify the gene we know the expression factor, why we want to know theexpression pattern, because this will allow us to expect some phenotype in the tissuewhere it is normally expressed.Then we move ahead for functional characterization of this gene, there are different approacheswhich is being taken care most commonly we are taking the gain of function approach andwe are trying to express a gene where normally it is not expressed and look what happens.This tells if a gene is sufficient for initiating any developmental program or not.Second approach which we take we take loss of function approaches, in this study whatwe try we try to silence a gene or knock out a gene or knock down a gene and then the lookwhat happens in a development, this allows us to understand if gene is necessary fora particular function.Then we move and we try to understand what is the mechanism behind a particular developmentalrole this is being done by studying the interaction of a particular gene, it can be genetic interactionphysical interaction with other regulator or its regulatory interactions.Another very important thing, so if we want to take a reverse genetics based approachas I said there are something which we have to fix first before identifying a gene firstthing what we want to fix is developmental processes.So, you have to define this is very similar what we have done in the forward geneticsas well.So, in forward genetics to identify a particular mutant or particular phenotype we fix thisprocess, here to identify a putative genes we will fix the process.So, if you are interested in looking for the meristem function or the genes which is responsiblefor organogenesis determinacy or developmental transition phase transitions.So, we will fix first the process, then you want to specify you would like to specifywhat kind of gene you want to study.So, there are some genes which are directly involved in some biological process for example,cell division or some enzymes which are basically controlling a fundamental processes and thensecond class of genes are regulatory genes.So, these genes are mostly regulating the activity of some of the developmental genes.And when it comes to the regulation there are different step of regulation gene regulationand at least major steps where you would like to look the regulation is transcriptionalregulation, here mostly we focus on the transcription factor specialized transcription factor.Post transcriptional regulation there are lot of developmental regulator which has beenidentified and they function after transcription like post transcriptional mostly they aresmall RNAs.Then there are known developmental regulators which regulate protein synthesis or translationof a particular messenger RNA, and then post translational regulation where you controlthe activity of a particular regulator by regulating or by modifying the protein throughpost translational modification.So, before starting any process we have to define this step by step we have to fix ourbiological process, we have to fix type of genes then we move and we try to identifykind of gene.So, we can take two approaches one is called candidate gene approach where you can identifyany single genes with a putative developmental functions.In second approach you can take more kind of global genes, which is called genomicshere you do not identify a single gene you identify a group of gene which are specificallypresent in a particular developmental processes and then you move ahead.So, what are the criteria which we look for identifying or for selecting a particulargenes which might have some function in the developmental processes.So, the there are two important parameter what we consider is first one is the homologybased analysis.So, first thing what happens that some of the model organisms their whole genomes aresequenced; if genomes are sequenced, then study has been made forward genetics basedstudies is quite advance and then you can identify some genes which has already beenshown for there some of the developmental function.And then you can look in other plant species, related plant species or in other class ofplant and then you try to identify if there is any homologous gene for that particularregulators.So, for example, some of the regulators they make a kind of gene family.So, there are multiple genes belongs to that family and sometime what happens the entirefamily is mostly regulating, some kind of developmental pathways or they are broadlyregulating multiple physiological and developmental processes.So, you can look gene family and then you can look the sequence similarity.So, if let us assume that you have one particular gene maybe in few slides I will show someof the example how this has been taken, but you can choose one gene which is known tohave some developmental function, then you can take the sequences and try to find outwhat are the homologous gene in another related species.And then if there is a common functional domains you can look for that and then another thingis that you see the relatedness which is called phylogenetic analysis.So, typically if a gene is having a high level of homology or if it is very related witha particular gene you can expect that it might have a similar function in other species.So, this is in silico approaches mostly bioinformatics approaches this will help you to define someof the genes which you might take for the functional study.Then second important thing you can check the expression pattern.So, this is very important because if you are interested let us assume you are interestedstudying some gene which is responsible for floral organ development, then you would liketo check the expression pattern and you will prefer a gene which might have a specificexpression in the flower or some of the organs of the flower.So, you can check the organ specific expression and then you can check tissue specific expressioneven within the organ if you want to define a very specific function.At the tissue level you can look for the expression pattern at the tissue level, if you are interestedmore kind of in the stage specific gene expression pattern you can look up a different stagesand then you can identify some genes which are present in one particular stage absentin another stage.And then you can for example, if you identify a gene which is not expressed in the vegetativestages, but it is expressed in the reproductive stages you may expect that this gene mighthave some function during the reproductive stages.So, these two major approaches we take to define a gene and you can see some of theexample, how these approaches has been taken.For example, in Arabidopsis thaliana a class of a transcription factor MADS box containingtranscription factor has been well studied and it has been identified that they playa very very important role in the floral organ development and floral organ patterning.So, this approach was used to identify some of the rice MADS box genes.For example, if you look this MADS 5, MADS 1 and they have been taken for reverse geneticsbased approaches to show their specific function during rice flower development.Similarly, so as I said that may be in some of the future classes we will study how flowerdevelopment is studied in model dicot Arabidopsis thaliana and there what we have found thatwe have found some classes of genes particularly A class, B class, C class.So, this is typically A class gene, then you have B class gene and C class gene and A classgenes has been shown to regulate sepal and petal identity B class genes are shown toregulate petal and stamen identity and C class genes are shown to regulate stamen and carpelidentity.And then if you look the sequence similarity homology functional domain in other speciesyou may identify the homologous gene.If I take some example for example, if you look this PI; PI is a typical B class genewhich interacts with AP3 to specify petal and stamen development in Arabidopsis.Now, there homologues like MADS2 and MADS4 has been identified based on the sequencesimilarity functional domain phylogenetic analysis in rice and when they have studied;they have been studied in rice, it was shown that actually they are involved in regulationof petal equivalent which is lodicule in rice and stamen development ok.But there is one interesting thing there might be lot of species specific variations as well.For example, there is only one PI gene in Arabidopsis, but rice the PI is duplicatedwe have two PI like genes MADS2 and MADS4 and when both of they were studied.Functionally it was found that MADS2 is more important for petal equivalent developmentwhereas, MADS4 probably is more regulating stamen development.So, this is one approaches where you can identify a putative genes to study.Then coming to the expression pattern analysis.So, expression pattern there are different ways to study expression pattern.Typically people used to do northern blot analysis in early days when genome or mostof the genomes were not sequenced.So, it was more kind of hybridization based technique maybe we will see some time later.But, what happens that if you see so, what we are doing that we are extracting totalRNA fractionating through the gel electrophoresis transferring on the membrane and then we areusing an antisense RNA or specific DNA or RNA probe which is radioactively labeled andthen probing with it.So, it will go and it will hybridize with the messenger RNA which is specific for thatprobe and then you can detect through the radioactive detection method.For example, if you look some examples, so this is one of the MADS gene and it has beenshown that this is leaf may be sepal, petal, stamen and carpel’s you can see that thisgene is very specifically expressed in the carpel, but it is not expressed in all othertissues.Similarly, if you look these are different genes and they are very broad very stronglyexpressed in the flower, but they do not express or they express very weak in leaf and othertissues.So, this tells a kind of; this is a kind of differential expression pattern.So, you can use the expression pattern, now we can expect that if I took this gene I canexpect that this gene might have some role in the carpel development whereas, this isa kind of general it has a flower specific expression.So, we can say that it might have some function in the flower.Then once you have defined a gene next question is how to isolate the gene?To isolate a gene if if your model organism or if your plant species which where you areinterested in, if the genome is not sequence then you have to take some approach to identifythe gene because you cannot design a primer you cannot do polymerase chain reaction, butif your genome is sequenced then it is relatively easy you can design a specific primer foryour gene your all the sequence are known and you can PCR amplify.But let us take first example if genome is not sequence, then one way of isolating geneis using cDNA library or genomic library.What are this library?So, basically what happens that if genome is not sequenced you can make their libraryand how you can do that; you can take either tissue specific cell or a particular organwhatever you are interested in you can extract total messenger RNA or total genomic DNA.If you want to make genomic DNA library, you will extract total genomic DNA, but if youwant to make messenger cDNA library you have to extract total messenger RNA here this willprovide the information of the expressed gene this will provide the information of the entiregenome at the DNA level.For genomic library you take the DNA do the partial digestion you can sheer it, you candigest it and then you can fractionate, you can choose what size or fragment you wantto generate library.Similarly, for messenger RNA you extract total RNA from total RNA you can isolate or youcan specifically fractionate the messenger RNA and then use this messenger RNA to generatethe cDNA synthesis.The typical way of generating cDNA synthesis is so, you know that a typical messenger RNAin case of eukaryotic they have poly A tail and you can design a primer which is oligodT.This oligo dT is a reverse primer which will bind to the poly A tail of all messenger RNAand then you can do reverse transcription process through which you can make DNA fromthe RNA.So, now your template is messenger RNA and you are use doing RT and then it will makea DNA.So, this is your DNA, so you can generate DNA from the messenger RNA take this cDNA,you can fractionate on gel and you can choose what size of library you want to prepare oryou can prepare all the library, and then you can use a suitable vector depending onthe size of your fragment or size of clones you want to generate, and then digest thisvector and do the ligation.Through ligation and transformation you can basically capture all the cDNAs; all the specificcDNAs which are present in a particular tissue or in a particular organ or in a particulardevelopmental stages and then you can clone them and put in form of different libraries.Now, since you have the library now you want to identify a specific gene from that library.So, there are two way of doing it depending on what kind of library you have made.So, one way of doing is colony hybridization and then plaque hybridization.Technology wise both the techniques are very similar what you do basically let us assumethat you have library here and what you can do, you can grow this colony’s on a plateand then you can do a replica plating.So, basically you can take a membrane and you can basically replicate or you can makea replica plate of this particular plate and then you use this membrane cell lyse and bakeit.So, that the DNA which are coming from this DNA, RNA anything which is coming from thisbacteria they are getting fixed or they are getting attached with the membrane, then takethis membrane and hybridize with your gene specific probe and then you can detect it.So, let us assume if you do the hybridization and detection if this is the colony whichis showing signal with your gene A using a probe against the gene A, then you can assumethat the corresponding bacteria or the corresponding colony might have a fragment of A. You isolatethis colony isolate DNA, isolate cDNA sequence and then you can identify your gene of interestfor your functional study.As I said if the genome is sequenced, then probably isolating gene is very easy, youcan do a simple PCR based gene amplification where you have to design gene specific primerand you can that you can design.So, basically if this is your gene you can design a short primers one primer which wecall forward primer, one as a reverse primer and then you can do a PCR amplification.Now, you can clone a gene this is called gene cloning, you can clone a gene and while youare cloning the gene you can either use the genomic copy or you can make a cDNA.Genomic copy basically, if you know and if you recall your basic molecular biology youknow that in case of eukaryotic genomes genes have exons as well as introns.So, if you use genomic DNA as a template and if you use a primer and amplify the gene thenyou are going to amplify both exon as well as intron; whereas, if you make cDNA for makingcDNA you are using mature messenger RNA and in mature messenger RNA all the introns arealready spliced out.So, if you use this method to amplify your gene then here you are going to amplify onlythe coding region which is spliced already spliced and this will be without the introns.So, depending on your use depending on the need you might choose what template you wantto use and this is the way you can basically identify or isolate a particular gene of interest.So, now you have defined your gene of interest where you want, which you want to study throughthe reverse genetics based approached.Now, next step was that you have already isolate the gene and then you can proceed for functionalcharacterization that we will take in some other class.Another approach which you can we are taking for doing multiple gene identification isthe genomics based approach.So, in genomic based approach what we do we fix our biological process our biologicalorgan or question and then identify at the global label what all are the genes whichare expressed or which are present there.So, genomics basically you can there are two classes of genomics structural genomics andfunctional genomics.In structural genomics what we do basically we genome we sequence the whole genome, thenwhen you sequence the whole genome you can use some of the already sequenced genome asa template and then you can do the genome organization.In genome organization you can basically identify what are the genes, what are the coding region,what are the non coding region.Then you can do genome annotation where you can predict the gene through using bioinformaticstool doing the homology based analysis, using template of other known sequences or referencegenome as a known sequences and then you can predict the putative function or you can predicta putative domain or class of the protein all these kind of things.Then another important thing what do we do in structural genomics is EST sequencing;EST is basically Expressed Sequence Tag.So, here what you make basically you can determine the partial sequences of some cDNA and thisyou can fix in a different tissue, different stages and different conditions.And this you can clone small fragment of the cDNA clone sequence to identify what are thegenes which are expressed in a particular condition.So, if you look this schematic diagram.So, you can do that if you have a genome sequence, you can do gene discovery, how you can doyou can take the in silico gene prediction, EST sequencing, full length cDNA which youcan do as I said either by PCR.So, there are other methods as well, then you can do a gene functional annotation, youcan do expression pattern analysis in silico prediction homology based then mutant analysiswe will look in other classes, in future classes, then protein-protein interaction.So, this is a way how to move for the functional; for the reverse genetics.Another genomics approach what we are taking is the functional genomic approach.So, here we first identify or we monitor gene expression and then we go for the functionalcharacterization and then trace out the mechanisms behind a particular process.Now, we will take one by one, if you have to start monitoring gene expression then firstthing what we you want to look that is there any organ specific gene expression or tissuespecific gene expression.Organ specific gene expression you can do; you can check in different organs this iscalled differential expression pattern, which is present in some organ absent in some organ.It can be organ, it can be stages, it can be tissue whatever you want and then you canidentify the differentially expressed genes or you will be more interested if expressionof a particular gene is developmentally regulated.If gene is expressed everywhere probably that might not be very regulated and then we wouldbe more interested in a gene which is specifically or present in a some condition, but absentin most of the condition.Then you can study the tissue specific expression; in tissue specific expression you can evengo and look the expression pattern in the organ; in the organ if you make anatomy ofthe organ do you want to look where exactly the gene is expressed which tissue it is expressedis more important.Then as I said that if you want to study developmental stages, so one development comes after thenext and then what you can do here you can identify the onset of a particular gene.So, let us assume a gene is expressed in a particular development stage, but it is absentin the previous developmental stage.So, you can go and identify what is the first developmental stage where this gene get activatedor start expressing this is called temporal expression pattern.Then we do promoter trapping how exactly we do we will discuss in another class.So, what are the different way which we are using in the plant developmental biology tomonitor gene expression as I said that first way and this is the old way what people weredoing was Northern blot analysis.So, quickly if I describe Northern blot analysis.So, we extract total RNA from two different organs, two different stages or two differentconditions, run them on gel and then we transfer the RNA on membrane and then you want to usea probe which is very specific for a particular gene.Hybridize the membrane and then check the expression and see how the expressions oramount of RNA varies across the tissues across the organs.So, these are some of the example if you look this has been done for some micro RNAs; microRNAs 393 and micro RNA 171 and if you look different tissues root, leaves, stem, inflorescenceand silique it was use.So, the probe was used against this micro RNA and if you look it has been tested ina wild type background as well as some of the mutant background.And you can clearly see that first thing the micro RNA is present in some of the tissue,it is present in the leaf, it is present in the stem, it is present in the inflorescenceand silique, but it is totally absent from the root.So, this tells the organ specific expression, so it is more kind of in the stem, in theshoot system it is present, but it is absent in the root system and then not only thatif you look these mutants.In mutants background the expression is getting reduced.So, for example, if you take the leaf case, so it is very highly present in case of wildtype background, but in mutant background the expression is reduced.Similar kind of things you can check here for other micro RNA.So, northern blot is being used here, but this is like as I said that previously itwas being used when advanced techniques technologies were not discovered because it involves akind of radio activity.Now though we have another method of non radio activity, but this is more kind of laboriousmethod.So, this is being used now in a very special cases, but this is not a kind of routine wayof checking expression pattern.Now, the more quick and more easy to do expression analysis is reverse transcription PCR.So, in reverse transcription PCR what basically you are doing.So, you are extracting total RNA, then you are using some primers reverse primer whichcan be oligo dT or it can be simply random primer and then you are doing reverse transcriptionto make cDNA and so if you have extracted total RNA from a particular tissue, then youwill convert all the messenger RNA into cDNA from that particular tissue.And then you can use some gene specific primer to do PCR amplification from that particularpool and then you can see whether a genes are expressed or not.For example if we look this gene these are the RT PCR product.So, you can see in a certain condition if you look in different tissues, these geneslook quite stable it is expressed in almost all tissues like root as well as leaves, butif you look the different conditions.For example, when you are inducing with the salt or inducing in osmotic, when you do notinduce the expression level is almost 0.But when you induce at different time point in root, then you can see that expressionof these genes are going up this you can monitor by RT PCR and this RT PCR is called semi quantitativeRT PCR.So, here what we are doing?You are making cDNA, then you are doing some round of PCR cycle and then you are takingthe end product of the PCR and running on the gel checking here.The drawback here is that you do not know after how many cycles the RT PCR will startgetting saturated.So, the now the alternate method which has come is quantitative RT PCR which is morekind of dye binding based assay here Syber green is very routinely being used and thiscase what exactly you can do, you can basically monitor expression pattern more quantitatively.And for example, if you look these are the different tissues has been taken, these aredifferent genes and here you can design a gene specific primer and you can monitor expressionpattern in the more quantitative manner for different genes in different conditions.So, this is another very important techniques which is being used for this one.So, we will stop here for this class and in next class we will take further of the functionalgenomics based approaches.Thank you very much.