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Classical Genetics Experiment

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So, we have been discussing about Mendelian genetics and we have seen many examples inwhich way understanding the genetic laws can really help us to do lot of genetic testing.In the same light I think is important for us to learn how new modern biotechnology toolshave started making huge difference in day to day medical applications you have justseen one video where you may have realized.That in which we were personalized medicine is not any more efficient if you understandan individual geno variation what kind of mutation might be happening in this particularindividual.So, then you can get some medicine which are very much a could be recognized by the receptorsof those mutations and then it will be much more effective for that individual as comparedto you are just giving some generic drugs to every individual.And therefore many times for a given treatment you may realize that you have lot of sideeffects and those side effects that just result of us we not knowing us exact way of modalityof the treatment.So, in molecular basis of inheritance we had discussed about some of the classical experimentsand then I will talk to you about some more classical experiment in which some of thefundamentals.That what is the genetic material in the DNA or the proteins were established and similarlymany of the transforming principles as well as how DNA replicates those kind of laws wellhow they were derived we are going to discuss about those experiments.Can a genetic trade be transferred into bacterial strains?There are two strains one is having capsule with S strain and one is not having capsulewith R strain.The one which is having S strain will may cause pneumonia other one is not going tocause pneumonia and this experiment was done by Griffith where he proved the principleof transformation, so we had seen the first condition.That if you have S capsule which you are now injecting in the mouse, the mouse dies becauseof the virulence from the capsule.If you have living R cells, then nonpathogenic they will not have any impact, so mouse remainhealthy.If you are having the heat- killed S cells killing the capsule layer, then that makesit nonpathogenic and again mouse will remain healthy.If you have lost situation like this when you are adding a mixture of heat killed Scells.And you are adding the living R cells what are the phenotype for the mouse, the mousedied.And that was because of, “Professor - student conversation starts” What happened in theexperiment the last one?The last one it gets killed because R cells acquires S cell traits.And how that happen?In which way R cell acquired the S cell characteristics?“Professor - student conversation ends.”So, in this case, probably some material, some DNA from the S cell got transferred toR cell and made them pathogenic.And this is what resulted in to this phenotype which was mouse died.So, what Griffith then concluded from this experiment that if we are injecting both nonpathogenic and pathogenic.But there is some kind of transformation is happening some substances is moving from theS cells to the R cells.And that is giving you the property which is making it much more pathogenic and creatingthe mouse to die.So, this kind of conclusion from this experiment.So, the living R bacteria got some substance from the heat killed S cells which is likea transformation is happening in this particular type of bacteria and therefore this particularphenotype was observed which is not so much common which was not expected.And that is where he first time realized that there might be some material could be transformedand that resulted in to Griffith transforming principle or the factors which are known asthe DNA or the transforming factors.So, now let us come to the next question which we started discussing briefly is protein orDNA the genetic material, And now who will explain this experiment?So, we have been saying that all the genetic based on DNA that DNA is going to transferfrom one to the next generation, right?But why by default we assumed that DNA is the only thing which is going to have thoseproperty which is going to get transmitted from one to the other generation.It might be even protein may have the same property, so somebody would have done it firsttime and thought about this type of this experiment right?And these are the two scientists.Hershey and Chase if you remember we discuss briefly that one could use some sort of labelsto find out or the track these kind of molecules in 1930s and 50s that time most of the experimentswere done very elegantly just by using some very basic regions and the labeling strategythen looking at things in the radio activity.So, if you know that you know you can label DNA because you have the phosphorus backbonein the DNA.With the 32 phosphorus you can label DNA, or you can label protein with some methionineresidues are there in (05:48) with the self for you can label proteins so they use thatproperty and let us kind of look briefly.So, just imagine they used one of the bacterial strain and now there is a virus which is phage.A phage which is if it can eat bacteria that is bacteriophage.So, now it is this particular virus is there so virus for its propagation transferred somegenetic material to the bacteria and therefore then it can have its multiple property.It can propagate in bacteria and make multiple copies of its own right.So, what are their thinking?Can we label both the DNA of it and if there is some DNA component of virus or even theprotein if you are labeling both of them.And now let us see what is going inside the bacteria which is going to create multiplicationso that will be probably one which is going to have all the genetic information so thatone could be extrapolate information format it whether the DNA or the protein have thegenetic information.So, to do this experiment they use a very simple technique, analytical instrument whichis known as centrifuge.In centrifuge I am sure you have seen washing machines, so something like washing machinewhere you are using a very high centrifugal force and let us say you have these are therotors in which you are keeping the tubes so let us say one of the tube is kept hereand when you are using very high centrifugal speed here.Let us say it know 10000 rpms etc. so then based on this particular speed If you havethis tube here whatever the bacterial membrane and the contents are there, they will justcome as a part of the bottom part of it which will be pellet and anything which is liquid,clear part that will remain as the supernatant.So, if whatever is going inside the bacteria along with bacterial membrane and everythingwill come inside which will become the part of the pellet.And anything which remains out will become the part of the supernatant that as the assumptionand he had labeled both protein and DNA.So, he was trying to see from radioactivity what is going inside the pellet and what isgoing in the supernatant part when he did this particular labeling experiment, whathe found that in the pellet.He could see radio activity of the DNA so based on the cell formation and whereas theprotein was found in the supernatant part.So, looking at this information he was able to conclude that the DNA is going inside thebacteria from the phage whereas the protein remains outside is not entering inside thebacteria and therefore protein and he can see in this supernatant part and DNA is comingin the pellet part.Just by doing the simple labling experiment he was able to conclude that the phage DNAentered the bacterial cells, but phage proteins did not.So, these are some very classical experiment done in 1900 that century which has resultedin to lot of fundamental information and if you think about it and I think these are notso difficult thing to do it just need some sort of concepts to be tried out.I am sure we have studied about chemical composition of DNA is you have been taught looking attwo DNA strand double helix model of DNA.Watson and Crick these are the scientists who illustrated the structure of DNA and theygot Nobel prize for it.Very briefly you have know 80 and GC base pairs whenever you have A, you will have theT with the two hydrogen bonds and G with the C3 triple hydrogen bonds.So, this kind of pairing will be there complimentary base pairing will be there.And now whenever a scientist actually Erwin Chargaff he derived the rule that a % A basesphere = % T base sphere whereas % G base sphere = % C base spheres and some of theother basic formation linked to the DNA.Now let us think about one of the properties of the DNA which was DNA replication let usthink about how DNA replication happens, so you are making multiple copies of DNA happeninginside the cell.DNA is double stranded, so people have proposed multiple hypothesis what are the possibleways in which DNA replication may happen?And they were have been three hypothesis proposed.Dispersive, semi-conservative and conservative.Semi-conservative has been most popular hypothesis.So let us look at this slightly in detail If you have this DNA, let us say dark bluecolor ones, both double helix Now in the first replication after that you can see one ofthe dark blue remain there and one of the new light chain new light form has appearedand now the second generation, one of the dark form and the light form remain thereand again light forms again is synthesized, which makes it again double helix.This is kind of DNA, semi-conservative replication the conservative replication is based on that1 DNA is always going to be like the original parental DNA, even in the after the firstand the second replication and the dispersive model says a mixture of both the strands ofthe DNA will be keep segregating after first replication and the second replication.So, again, these are the hypothesis.Now people were kind of puzzled at what is the way of DNA replication to happen and ascientist, a group of scientists in fact Meselson and Stahl.They did a very nice experiment and try to prove in which way the DNA replication mayhappen so let us assume that we have this DNA in the parental strand.And both of them are N15 labeled after the first generation one of this strand from theparental one remains there and a new form which is from the light, so this is N15.One is strand comes from N15 and 1 Meselson size N 14 and from this one we have one ofthe blue one and one of the white.And from this again alright same will happen again here as well right.From this part okay now let us think about the % of.How much we have heavy farm here in the parental 1 for N 15 so that is 100% right.Now if I have grown N15 DNA in a medium which is light medium and 14 medium.Now the second strand of DNA is N14.What is the ratio here for the hybrid?It is a Hybrid.The hybrid means it is having both N14 and N15 mixture of that right.So, in this particular one, in the first after the first replication everything becomes hybrid100% hybrid forms here.Now as it goes to the second replication how much is hybrid and how much is the N14?This is hybrid, right?This is a hybrid right so these two are as well as these two these are part of the secondreplication so what is the %age of hybrid and %age of N14?50 - 50 right.So, there are ways of doing the density centrifugation where you can separate these based on thedensity.They use cesium chloride based density to separate this particular bands.Light or heavy form of band and this is how the experiment was being done, so initiallythey grow bacteria in and 15 are the heaviest isotopic forms.Then they transferred that to the light form or the N14 medium and now from there theycan see the bands you know is that coming in the center or there is some new brand appearingwhich are less dense than that and when they analyze those particular fractions.Then they are able to conclude what is the ratio of N 15, what is the ratio of N14 andN 15 and what is the from 1 to next generation, how much % they can see the change.So if you see in this particular experiment here in the hybrid band, which is having both100% after the both replication and after second replication you have 50 % of the hybrid bandand 50 % of the light band appearing.Now if you go to the next generation what will happen?Please draw 25 75 somebody mentioned rightly alright.So, this is pretty much a way for them to prove that among the three models which areknown are dispersive model.Semi conservative conductor, even conservative probably the DNA replication happens usingsemi conservative model and just by growing DNA from N15 and N14 medium and analyzingthe DNA contents they were able to make this conclusion.Let me explain more detail in the following animation.(Audio from 16:04 to 19:38) According to the conservative model, the two parental strandsof DNA as a whole, serve as a template for the synthesis of progenerity and A moleculesthus one of the daughter DNA molecules is actually the parental DNA while the otherdaughter DNA consists of two newly synthesized strand from fresh nucleotides.The dispersive model of DNA replication hypothesizes.That the parental DNA molecule is cleaved into smaller double stranded DNA segments.Which serve as a template for synthesis of new DNA strands.The segments then reassembled into completed DNA double helix with parental and daughterDNA segments interspersed.The content of parental DNA is a double helix goes on decreasing with each generation.According to the semi conservative model of replication.Each parental strand act as a template for the synthesis of a new strand of DNA whichis complimentary to the parental strand.Each daughter DNA molecule always has 1 parental DNA strand and one newly synthesized daughterstrand.Of the three replication models suggested Meselson and Stahl proved that the semi conservativemodern was correct for this.They grew E coli culture for several generations in 15 containing medium.So, that the basis in DNA contained N15 instead of N14.Next and they transferred and grew the cultures for several generation in and N14 containingmedium.Throughout the period of growth samples were taken cells listed the DNA analyzed.By centrifugation and in CSCL gradient.The parent DNA showed 1 band in CSCL gradient corresponding to N15 DNA.The first generation daughter molecules also showed 1 band which was not at the same positionas parent DNA.This correspondent to N14 N15 DNA while the second generation showed two bands.One of N14 N 15 and the other of N 14 light DNA.These results exactly matched the semi conservative replication model.So, this brings the end off this entire section on genetics, Are you now familiar with Mendelianlaw based on Mendels experiment on the classical pea plant.You now also know how a Morgan independently tested A Mendels observation, in mendel experimentusing another model system which was drosophila melanogaster or the fruit fly which providedthe evidence that the chromosomes are indeed the location of Mendels heritable factors.Then we have discussed about the classical genetics experiment which proved that theDNA is heritable factor and it is transferred across generations.You are also introduced to the concepts of genetic recombination and linkage betweengenes and how it affects the inheritance of characters.We then discussed about few chromosomal abnormalities and looked at you know various examples andsyndromes.How alteration of chromosome numbers and even structure could cause some of the geneticdisorders.In the next lecture we will start about you know a new topic thinking about the bacteriaand other prokaryotes and then we will talk about some of the applications which are linkedto those microorganisms.Thank you.