00:05okay so we still we have a few people
00:07trickling in i'll just um
00:10i'll do what i've been doing instead of
00:11the poll i'm just taking the screenshot
00:13of the participant list after it looks
00:16all the activity has stopped that's a
00:19little easier for me to
00:20reconcile than doing the poll but we
00:24important goals today we want to first
00:29finish our dog paper and so i went back
00:31through i couldn't remember exactly
00:32the precise point where we stopped so
00:35i went back and looked at the points i
00:37wanted to make in the second half of the
00:38paper i know we finished the
00:40quantitative pcr stuff at least the
00:43technique not the results
00:45uh so i have some notes on that i want
00:46to go over and then after we're finished
00:50uh do some review if you guys have
00:52questions about the the material
00:54and uh and make that the the remainder
00:58then on monday of course the exam will
01:01happen so we won't have a normal class
01:03and then on wednesday we'll start back
01:05what's next oh the next paper is about
01:10the bears so that one's that paper is
01:13actually pretty straightforward and it
01:15a pcr riff flip so it'll reiterate
01:18something that we've already
01:20learned and the point of that paper is
01:21more about hardy-weinberg and population
01:25than than about the science part the
01:28science part is just how they get the
01:29data then they analyze it and that's the
01:30part i want to focus on with the bear
01:35many of you may remember having taken
01:39that we used some of that data when we
01:40talked about hardy weinberg but of
01:42course in this case i want to go a
01:44deep than we did in that class
01:48okay so let's get right back into the
01:52waste any more time me talking about
01:58okay let me get the chat up and i am
02:01recording yes all right
02:12okay so if we go right back into the
02:17results and discussion um
02:20i think we may have started this but
02:22even if we hadn't i went back and
02:26highlighted some things that i want to
02:27bring out because after looking at the
02:29review and i'm starting to
02:31put some of the finishing touches on the
02:34um i want to make sure that we don't
02:38uh to bring us back to where we were we
02:41identified three different
02:42mlph haplotypes recall that a haplotype
02:46group of genetic variations that are
02:48inherited uh as a unit
02:51okay so we'll look at their haple types
02:52which are pretty extensive in just a
02:55well we can sort of see them at the
02:56bottom of the screen there in figure one
02:59haplotypes are these long
03:02groups of variations that um are
03:06okay so the three digital haplotypes did
03:09any variant alleles in the coding region
03:13all of the variation detected up to the
03:16point where they began
03:17the bulk of the experiments for this
03:20uh did not show any causative effect so
03:23and they were expected there there to be
03:26some variation in the coding region
03:27there was not so the most obvious
03:31explanations are not going to be what's
03:36but they did have they did notice that
03:38in the variations they were looking at
03:40there were similarities in the
03:44of the dilute haplotypes the the
03:46haplotypes from the dogs that had the
03:48the common dilute um phenotype so
03:50they're assuming that there may be
03:53in this five prime five prime region
03:55which is not part of the coding region
03:58but is important and so uh that they say
04:03said there regulatory mutation might be
04:05responsible if this was true
04:07it would be expected that the mutation
04:09will be located in a region where the
04:10three haplotypes share common alleles
04:13okay so from the dilute dogs from the
04:16the group that they looked at
04:18all those dilute dogs could be put into
04:20one of three categories that's what
04:22they're calling these three
04:24common haplotypes okay so at this point
04:26all of the dilute dogs do not have a
04:29but they fall into one of three groups
04:32what they're proposing
04:34is that there may be within this cluster
04:38a commonality that can be explained
04:41as the cause of mutation for all the
04:43dilute dogs so in other words
04:45they've narrowed it down to three
04:46haplotypes they want to know within
04:49what's common and what's not okay so
04:53they choose six dilute and six wild-type
04:54dogs they have a much much larger panel
04:58of dogs but they want to start small and
05:00see if they can find something and then
05:01like in the sabino and some of the other
05:05papers we've looked at once they find
05:06their their proposed cause of mutation
05:09then they can extend it to the bigger
05:11but for the beginning they're just going
05:12to choose six of each group
05:15and remember they can figure out the
05:18based on pedigrees as well as for the
05:21they're all homozygous recessive okay so
05:26and this is an impressive uh
05:29amount of dna that they are they're
05:30producing from pcr and then sequencing
05:33especially at the time so this was in
05:36sequencing was fairly well uh you know
05:39streamlined by then but this is still an
05:41impressive amount of work
05:42so 11 905 base pairs so of the the
05:45region that they wanted to get
05:48they were able to acquire 95 of it so
05:5611 161 or 89 could be accurately
06:02so sequencing reaction even though
06:05sequencing reactions even though they're
06:08can produce errors and if you're looking
06:10for a single nucleotide polymorphism
06:12then one error in your reaction you know
06:15you could be on a wild goose chase
06:17thinking that was the cause when in fact
06:19it was just an experimental error and
06:23is uh notorious for introducing
06:26uh at a small rate so that's why you you
06:30re-sequence and that's why when they did
06:32the human genome project and other
06:33sequencing projects they would say they
06:41um i've been lots of messages about
06:44people late because of
06:46evolution uh dr lydiard
06:49why is he doing this to me and what can
06:53nothing he's my boss and he's like a
06:58um okay well that's okay you guys can
07:00watch the beginning on the recording and
07:02catch up as you can so i didn't take a
07:05attendance uh by screenshot here in a
07:09okay so there are a couple things in
07:11this paragraph that i made a little
07:12note to myself one was um the missing 11
07:16were mainly due to an extreme gc content
07:19especially within the proximal promoter
07:21so there's actually a lot in this
07:23sentence even though you could just read
07:25right through it and not
07:26uh you know maybe no alarms go off but
07:29it's really an important statement so
07:31an extreme gc content especially within
07:34the proximal promoter
07:36proximal promoter is a reference to
07:39the region immediately upstream of the
07:41transcriptional start site so promoters
07:44could span thousands of bases
07:45potentially and have lots of regulatory
07:48but the proximal promoter that proximal
07:52you can see is a word related to
07:55which means next to so the proximal
07:58promoter region is the part right by the
08:00transcriptional start site right at the
08:02and if you look in the figure they've
08:05actually made a point of noting
08:07the gc content down here at the at the
08:11and the gc content spikes right below
08:14the transcriptional start site so the
08:16arrow remember indicates the beginning
08:19and that's where the gc content is
08:22if you look at the the legend eighty
08:24percent so that's a lot of g's
08:26and c's that's way above what you would
08:30average region of dna so there are a
08:32couple things about that first
08:36they're saying that the missing sequence
08:38data is from this region because of the
08:41and the gc content does influence how
08:45uh dna can be sequenced one of the
08:48content and then i also have another
08:52note about cpg so i'll address both of
08:55out of our screen share for a minute so
08:57one of the problems with
08:58with sequencing is when you uh put the
09:01dna into the sequencing gel
09:02it is single stranded okay so you make a
09:06um if you recall you have our template
09:11dna and then you produce your sequencing
09:15that sequencing sample will terminate
09:17you know because of the dye
09:26labeled single-stranded dna is what goes
09:29and so that goes into the gel or if it's
09:32high throughput sequencer into the
09:36but whatever it is that you're using to
09:38look at the length of this dna it is
09:41one of the issues that arises when you
09:43have lots of g's and c's
09:49that has high gc content
09:52is hard to keep single stranded because
09:54this guy that has all these g's and c's
10:01will have a strong tendency
10:05to form regions of base pairing with
10:09so it'll form these loops like this and
10:10create these secondary structures
10:13the the c's and g's are just too
10:15compelled to come together in base pair
10:17now a c and a g you know scattered among
10:21isn't going to create this but when you
10:23have them in long runs like this
10:25they tend to do that and there's a
10:27reason that they tend to
10:28occur like this at proximal promoters so
10:31the first thing is we have the secondary
10:34that create issues for sequencing the
10:36second is why are they at the proximal
10:39that's the second point i want to make
10:41because they spike there
10:43so the proximal promoter if we're
10:46looking at a region of dna and here's
10:49transcriptional start site the proximal
10:52promoter is the region immediately
10:58and this is a region that you would want
10:59to regulate if you want to turn genes on
11:02one of the ways that genes are turned on
11:05is by methylating the dna near the
11:08promoter so that proteins
11:10and specifically transcription factors
11:12can't bind so methylated dna
11:18will shut down transcription so it's
11:21common because of this
11:23to have c c's and g's at high
11:26uh percentages in the vicinity of the
11:28promoter so that the gene can be turned
11:30on and off by these regulatory
11:32and when c's and g's occur like this in
11:36in succession there's a name for that
11:40and we'll have a few times this semester
11:42where we look at these things
11:44but they're called cpg islands so
11:47they're regions of dna with high
11:50cg repeats the p is a reference to the
11:52phosphate between them so
11:54it's an island in the dna like here for
11:58where there are lots of c phosphate g
12:02uh individual units repeated over and
12:04over again so they call it a cpg island
12:07it occurs in just a little region on a
12:10promoter or elsewhere in the dna
12:13so that's an important thing because it
12:15could it could be important in why this
12:18dna is behaving as it is but
12:21if not even if it has nothing to do with
12:24the dna itself it sure is causing
12:26problems for the sequencing so
12:28i guess a couple different things going
12:32okay back to business here so
12:34comparative sequencing revealed
12:3673 polymorphisms so there's a lot of
12:38variation in this region
12:40which is kind of unusual anyway because
12:42it's you know you would think there'd be
12:44a lot of conservation
12:45in the regulatory area but we've all
12:48learned what these are now
12:49microsatellites indels and 63 snips
12:54similar to their study before three
12:56different dilute associated haplotypes
12:58were observed so this becomes
12:59consistent they call it d1 d2 and d3
13:04and the haplotype uh that they found
13:08that d1 d2 and d3 are again they're all
13:11closely related so they're
13:12sort of grouping this together but
13:16um the third one is interesting the
13:19third dilute haplotype is called d1 i
13:21don't know why they didn't call
13:24never mind anyway the d1 they call an
13:28um represents the ancestral haplotype on
13:32which the hypothetical
13:33founder mutation occurred
13:36and this is another term that i don't
13:38know if we saw it in
13:40the previous papers or not but i know it
13:42will come up during the semester
13:45more than once but a founder mutation is
13:47thought to be the first instance
13:50of a mutation causing a phenotype so we
13:53actually look at this in detail in this
13:54class but an example of this
13:56would be the short-legged mutation that
13:59occurs in a lot of dog breeds
14:01when they looked at the cause of
14:03short-leggedness which is
14:05i think it's called chondrodysplasia in
14:07dogs but when they looked at the cause
14:09it it actually could be traced back to a
14:13so i believe if i'm not my memory is not
14:17all of the dogs and all of the breeds
14:19that they looked at that had short legs
14:21had the same mutation meaning that at
14:23there was one original occurrence of
14:25that and then it spread to all of the
14:27descendants and was bred into different
14:30so in this case because they are
14:32observing similarities among these
14:34different haplotypes
14:35they are proposing that there is a
14:39which is going to turn out to be the
14:40common cause of dilute
14:44phenotypes among all these animals so
14:45we'll see here in a minute how that
14:49but that's what they mean by a founder
14:50mutation the original one if you go back
14:54that is responsible for all of these all
14:56right so the comparison between
14:58the dilute uh associated haplotypes
15:02uh revealed 14 polymorphic sites
15:04perfectly associated to the dilute
15:07dilute allele not allele and so those um
15:12well let me go on and then i'll show you
15:14the figure the most interesting among
15:17was an ag snip located at the last
15:19nucleotide of the untranslated
15:21exon one so in this particular
15:25gene we look here this particular gene
15:29exon is transcribed and it's spliced and
15:34but the actual start codon doesn't occur
15:37i believe exon 2. and so when they
15:40looked at the coding regions previously
15:42they did not include
15:43exon 1 in their analysis because that is
15:46the start codon that begins the sequence
15:51however exon 1 is still part of the gene
15:53right it just isn't part
15:55the part that encodes the amino acids so
15:59they found a mutation
16:02among all of the ones that they were
16:04looking at the 14 different ones
16:06that potentially had a cause because
16:08it's one that would have a functional
16:10importance to the processing of the
16:12it's right at the splice junction
16:14between exon and intron
16:16when they discovered this they went back
16:18and looked at all of the 285 dogs in the
16:21larger panel that they had
16:23and they found again perfect association
16:26between this snip and the dilute
16:29phenotype so here's where it occurs
16:33get my pencil go away
16:36so here's the mutation right there
16:39and this is um this is not a contiguous
16:44sequence at first i found this diagram
16:47because i looked at this mutation and i
16:49saw what they were calling the splice
16:51it's not a splice sequence so that
16:53didn't make any sense but then i
16:54looked once i got to know the paper a
16:57little better i looked down here and
16:59actually is showing the location where
17:01all of these nucleotides take place so
17:03if you notice they are not
17:05in sequence one after the other they're
17:08sequential and that they occur in order
17:11for example these two snips are
17:16201 bases apart so this a
17:20occurs 201 bases before this t and the
17:23reason there's that much space is
17:26in between those two sites everything is
17:29okay so you have there's no reason to
17:31put all 11 000 bases out there
17:34all they need to do is show the places
17:35where nucleotides are distinct
17:38where they form the haplotype and so
17:41perfectly associated in all the dilute
17:43dogs and all the wild
17:45dogs that nucleotide position
17:48is a g okay so let's let this play out
17:52in a previous study they analyzed only
17:54coding regions so we talked about that
17:56and no single polymorphism was
17:57identified in the coding sequence
17:59so that was of course the first mystery
18:01in the first disappointment
18:03so they went back now and they found a
18:05mutation that's in the messenger rna
18:07but not in the coding sequence so why
18:12well if the intron is not correctly
18:15spliced out this could create problems
18:19the messenger rna that gets read by the
18:21ribosome even though it precedes
18:24uh the coding region so there's there's
18:26a reason for that that's on the next
18:27page but let's look at their analysis
18:30so in uh at the sp so here is the actual
18:34splice site sequence so i
18:36scribble with my five-year-old
18:38handwriting there the end of exon one in
18:40the beginning of intron 1.
18:42the consensus side i think we've talked
18:43about splice consensus sites before
18:47would be c-a-g-g-t and the splice
18:51event occurs between the two g's in the
18:5584 percent of the time and this is based
19:00157 640 splice sites
19:03based on that study 84 of the time that
19:08where this polymorphism occurs is a g
19:11so that's a pretty strong uh pretty
19:14strong evidence that there's
19:15conservation and there's an importance
19:17that nucleotide so if it were to change
19:20that could really throw throw things off
19:23and that is exactly what they are
19:25hypothesizing so if the splicing of
19:28intron 1 is not correctly executed
19:32it seems likely that any apparently
19:34spliced transcripts would contain
19:36premature stop codons and be subject to
19:44all right so this is why they think it
19:48and i think that they present a good
19:51present good support for it nonsense
19:54mediated decay i think that's on our
19:57review sheet as an exam topic
20:05nonsense mediated decay which is usually
20:11is a way for a cell to not
20:15mistakenly translate messenger rnas that
20:19have premature stop codons
20:21if they did they would produce protein
20:23products that of course were
20:24were non-functional and potentially
20:27scientists it's not been too long ago
20:30that the scientists discovered this
20:31but there's actually a system in cells
20:35messenger rnas that have premature stock
20:37codons i believe the original
20:38observation that led to
20:40to this discovery was that people looked
20:42at messenger rna levels
20:44of wild-type transcripts and messenger
20:50transcripts that had premature stop
20:51codons and they recognized that there
20:56the number of messenger rnas with the
20:57premature stop codons should have been
21:00right because the mutation doesn't cause
21:02the rna to not be expressed it just
21:04causes a problem in the protein
21:06what they discovered though was that
21:07there was a startling difference
21:10in the abundance of wild type rnas
21:12versus rnas that have
21:14premature stop codons so they
21:16hypothesize there must be something in
21:19that knows to get rid of those messenger
21:20rnas specifically and so that thing
21:22is called nonsense mediated decay and
21:26is that after messenger rna
21:30is spliced and is ready to be translated
21:34the junctions between exons the
21:37so-called exon exon junction complexes
21:42have proteins that hang around after
21:46so these are called are the specifically
21:49the protein complexes
21:50are called exon exon
22:04so after splicing takes place some of
22:06the proteins involved in that splice
22:08stay at the site okay and then when the
22:11ribosome comes along
22:16and starts reading this messenger rna
22:20exon exon junctions let's see how much
22:23have to work with here
22:27those exon exon junction complexes are
22:30removed so the ribosome
22:32cruises down this messenger rna i'm not
22:34doing very good with my
22:35scale drawings there and produces this
22:38protein and so this is the finish
22:41that's obviously that's a protein
22:44uh so as it goes down the exon exon
22:48complexes all those little proteins get
22:52tossed off of the messenger rna so from
22:54this point forward this messenger rna
22:57is clean of proteins other than the
23:00ribosomes that are reading it
23:02okay now if there was a let's say that
23:04there was a mutation
23:07a premature stop codon
23:11so we have our complexes here if there
23:14was a premature stop codon
23:19then that means our ribosomes would
23:22this point right the ribosomes would get
23:24to here and then they would
23:26dissociate so what does that mean for
23:30it means that during the process of
23:32translation after this has been
23:34exported to the cytoplasm and ribosomes
23:36are trying to translate it
23:38it persists to have these exon exon
23:42if those aren't removed this messenger
23:46will be degraded by nonsense mediated
23:51so if the exon junction complexes stay
23:54on the messenger rna
23:55proteins associated with nonsense
23:58will attack and degrade those rnas
24:01they'll leave these rnas alone so it's
24:04kind of an interesting system obviously
24:06i'm glossing over some of the
24:08the finer details of which proteins are
24:10involved and so forth but it gets a
24:13uh crazy after a while but the point is
24:17and so they're proposing in this paper
24:20that this is the reason
24:22why having a mutation in a section of
24:26a section of the gene that isn't coding
24:30matters and so if those transcripts are
24:37destroyed because of the the instance of
24:40stop codons because of this splicing
24:42then the melanophilian gene even if the
24:45coding region is intact
24:46is not going to make it into the you
24:49know it's not going to produce
24:50proteins so that i think that's kind of
24:54and they do give a few numbers here so
24:57analyze the splice site mutation using a
25:00computer program that
25:01you know guesses that it's not guesses
25:03predicts its efficiency
25:05and they say it goes from 99 percent
25:10they do mention something else here
25:13again in our our goal here is to learn
25:16as much as we can from each of these
25:17papers so here's another little
25:19side note that might have caught your
25:20attention and maybe you already know
25:24but the the analysis they did predicted
25:26that the polymorphism the mutation
25:32to 12 percent additionally it
25:36the mutation completely abolishes the
25:39accessory splicing factor
25:42binding site so what are the two the two
25:44things you're talking about here
25:50is in this accessory splicing factor is
25:54that acronym stands for sometimes it's
25:56also called alternative splicing factor
25:58but the u1 and the ssf or sorry sf2
26:05we don't talk a lot about snurps but we
26:07should mention what they are here
26:19which begs the question what is a snurp
26:22and a snurp is a small
26:26nuclear ribonucleoprotein
26:33small nuclear ribonucleoprotein these
26:37make up the uh spliceosome so
26:46u4 u5 and u6 i think that's right i
26:50wrote that down because i was afraid i'd
26:52i don't know what happened to three so
26:54collectively these guys make up
26:57the spliceosome and i think you remember
27:00the spliceosome is the complex of rnas
27:04that um that facilitates splicing of
27:09so the u1 snip specifically
27:13binds by base pairing
27:18let's see how do we spell base pairing
27:23to the five prime splice site
27:29so because a snurp contains rna and
27:32complexes within its structure it can
27:36with with things so the u1 snark base
27:39pairs with the five prime splice site
27:41which is the thing that is mutated
27:43on this gene so if it's mutated it's not
27:47and then the other protein which was oh
27:50look at my paper here instead of screen
27:52asf that's right the other protein asf
27:56also known as sf2 that's the same
27:58protein just different names
28:13to recognize and splice
28:18i'll just actually the better word would
28:22it's also essential to recognize and
28:23spice splice slash cleave
28:26at the five prime supply cipher
28:32okay so both u1 and ss sf2 asf
28:36are both essential for recognizing the
28:39five prime site and causing the cleavage
28:42at that location both of them are
28:45uh much much diminished activity
28:49because of this change okay so we're
28:53pretty much at the end here just a
28:54couple more things to say
28:56and then we'll be done with our paper so
28:59that because of these because of this
29:02mutation and because of the effect it is
29:04on splicing efficiency
29:08recognition by u1 recognition by asf
29:11chance for functional transcripts
29:15with the mutation are going to be very
29:18okay if their predictions are true
29:22then the messenger rna levels in
29:25melanocytes should be
29:26should be quite low so they take skin
29:29from these dogs kind of sad but they
29:32take skin biopsies and they
29:35do quantitative pcr and that's what data
29:39diagram here the in general they get a
29:44fuzzy on their precision here but in
29:47d which are wild type dogs showed the
29:49highest homozygous dilute dogs showed
29:51the lowest expression
29:52heterozygous dogs were at an
29:55and i want to come back to
29:58this little detail heterozygous dogs at
30:01an intermediate level in a second
30:04okay so on average the dilute dogs had
30:08of the transcript compared with the
30:09others and i'll read the conclusion here
30:12before we look at the graph
30:13now at the time it can't be at this time
30:15it can't be formally proved that this is
30:19they mentioned that there are sequences
30:21in that interval that they just
30:22couldn't effectively sequence yet
30:24because of the gc issues that we talked
30:27so they can't exclude the possibility
30:29that the other polymorphisms remember
30:32based on their hypothesis that it caused
30:34a splicing problem but that doesn't mean
30:36the other ones aren't also important
30:38but i think that they do make a good
30:40case the combination of genetic data
30:42preliminary analysis and the position
30:44strongly argue for this being a
30:48so the data if you look at it here just
30:55it does show the trend so the squares
30:58the the x's are adults uh what we can
31:01look at to make life a little simpler is
31:05and so as we go from the wild type
31:08to the dilute dog over here the average
31:13expression does go down again they could
31:15do better by having a lot more
31:16data points here but it's the trend is
31:20and so i think it's it's definitely i
31:22think it's valid it's just
31:24not ideal but the last thing i want to
31:26say about the paper and then we we can
31:29this statement here heterozygous dogs
31:31are found at an intermediate level
31:33i think this is interesting because
31:37have the wild-type phenotype so this is
31:43on in terms of mendelian genetics this
31:46is clearly autosomal
31:48recessive no doubt about it all the
31:52pedigree information tells us that about
31:55i think it's one of the first thing they
31:57say in the abstract is autosomal
32:00but if you didn't know that already and
32:02i just showed you this graph i think you
32:05be forgiven for making the mistake that
32:09incompletely dominant trait remember
32:11incomplete dominance is when the
32:13heterozygote is intermediate between the
32:16so i think this is a good example where
32:19gene expression sometimes operates at
32:22so if you have a certain amount
32:26you know having in this case the
32:27melanophil and rna between here and here
32:29you're fine but if you dip way down here
32:34so even though the intermediate the
32:35heterozygote doesn't have the full
32:37expression of the homozygote
32:39it has exactly the same phenotype
32:42and so you can be mendelian on you know
32:46but if we dig down to the molecular
32:48level it looks like it's incompletely
32:50dominant it just doesn't
32:51manifest that way so it's just just a
32:54word of warning and when you
32:56interpret data like this it's good to
32:58know what's happening in the whole
33:00as well as what's happening at the
33:04okay so that is all i want to say
33:08about our dogs so i will
33:11stop my screen share there and ask if uh
33:14folks have questions and now that we
33:19everyone here i might take a quick i'll
33:21just take a screenshot of our
33:23participant list and that will help me
33:26check attendance later although i think
33:2940 maybe all of us may be wasting my
33:34wouldn't be the first time i've wasted
33:36my time doing something
33:38and it won't be the last
33:42it's a campaign pledge i tend to keep
33:46okay any questions that you may have
33:51while you're thinking about uh the exam
33:55i can't tell you the number of questions
33:57that are on the exam yet because i
33:58haven't finished it i've been
34:00working on it but i've got some i've got
34:02a ways to go unfortunately
34:07at least it's not sunny and beautiful
34:08outside so i won't want to
34:10get away from my desk
34:22i cut my hair especially for this review
34:35yes deanna i love the hand raising hi
34:40the um sorry i'm looking at the
34:44question the genetic heterogeneity
34:48in sabino phenotops is that just
34:49referring to the fact that
34:51it was sabino and then tobiano and
34:55like different um genes could play a
34:59different role if there's like a
35:00completely white horse and
35:06blanking on how to word this
35:10i'm just letting you suffer through it
35:12because i do that all the time i can't
35:14the words are swirling i can't get them
35:16to come out straight
35:17i can address that question if you want
35:21if you unless you want to keep trying to
35:28is genetic heterogeneity uh just plagues
35:32people sometimes because it's such a i'm
35:35why really i don't know that it's a hard
35:37concept but it's a weird concept
35:39in the case of the sabino phenotype
35:40there i guess there are a few things
35:44is that if well if if i for example went
35:47into a field of horses
35:49and they had white and dark patterns
35:52i wouldn't be able to even tell between
35:54sabino and tobiano and overall
35:57lethal white syndrome patterns but some
36:00people probably could if you've seen
36:01enough of them i think
36:03there is actually a clear distinction
36:04between those three and so those are
36:06actually different phenotypes i believe
36:09that's accurate to say that for coming
36:12from a non-horse person that i am
36:13but i believe it's accurate to say that
36:16the tobiano is not sabino although it's
36:19and the ovaro is not sabino although
36:22but in the case of the the specific
36:24question i'm asking there
36:26at the end of that paper they they
36:28determined that the gene that they had
36:31discovered and the mutation in that gene
36:33which was kit well they didn't discover
36:35the mutation they found in the kit gene
36:40sabino horses that they looked at was
36:43perfectly associated with the
36:45with the phenotype then they expanded
36:48their analysis and they found that
36:50sabino was caused in other cases
36:53by a different mutation because they
36:55found that there was a group of horses
36:58who had the wild-type allele for
37:02the kit mutation they had discovered but
37:05still had the phenotype
37:06and so the genetic heterogeneity this is
37:08why they called it sb1
37:10they are assuming well it's not an
37:12assumption it's it's a
37:14true observation that there's more than
37:17mutation potentially in more than one
37:21that is causing the sabino phenotype
37:23they believe they found one it accounts
37:25for a large group of sabino
37:27mutants and it does so accurately then
37:30they predict there's another group that
37:31must be caused by either a different
37:33allele or a different gene
37:35so i guess potentially it could be a
37:36lilac heterogeneity because i don't know
37:40that it's not in elsewhere in the same
37:43but what they do know is they found one
37:46cause of the sabino phenotype
37:48but there's also another one that has
37:49yet to be discovered maybe it has been
37:51discovered by now actually i haven't
37:53um but that's what it means they have
37:56discovered one cause
37:57but they know that there's another one
37:59out there that is different than the one
38:03let's see um here's a question
38:08could you talk about how rtpcr revealed
38:11second one the second one is related to
38:18r r t p c r r r t p c r sounds like a
38:22and q pcr the same thing i've confused
38:24myself you're not the first
38:26uh so it is confusing especially because
38:31rtpcr is an acronym that has that could
38:34mean two different things
38:36so i'll um let me address the second
38:39question first because it's quicker so
38:41if somebody says they did rt pcr
38:44you need to ask them what the r and the
38:48stand for because rt pcr can be
38:53reverse transcriptase pcr
38:59which means you take messenger rna you
39:02produce c dna from it and then you can
39:04sequence that cdna and see
39:06you know see what you find rt pcr
39:10can also be and is also a reference to
39:14real-time pcr real-time pcr
39:22as quantitative pcr or qpcr
39:25so in the case of real-time pcr this is
39:27when you do a pcr reaction and you have
39:29the k the type we talked about was the
39:31tacman probe that's not the only type
39:33but it's the only type i'm going to ask
39:35you about in real time you can watch
39:38the emergence of messenger rna levels
39:41well actually let's be clear you're
39:43watching the emergence of
39:45cdna levels but it is
39:49relative to the original messenger rna
39:51levels that you started with which you
39:52converted to cdna and now you're doing
39:56so you're watching these levels climb in
39:58real time with your different samples
40:00so that's why it's called real-time pcr
40:02because you can stop at any moment and
40:05how much dna was present at that time
40:07and then you can infer
40:09well there must have been such and such
40:10amount of messenger rna
40:12quantitative pcr i think was a term
40:15later to distinguish between these two
40:18most of the time today people would say
40:23but originally it was called real time
40:25it was just an unfortunate
40:27accident that not an accident it's just
40:29an oversight that they didn't
40:30start out with a better name but it's
40:32just unfortunate that they happen to
40:33have the same abbreviations
40:35so we talked about real-time pcr with
40:41dogs we talked about reverse
40:45with the sabino horses and so this goes
40:48back to the previous question could you
40:51rt pcr revealed the defects
40:55and so in the case of the sabino horses
40:58let's see what figure that was okay so
41:06it's figure two i think i've got it
41:08here i can pull it up for you in the
41:10case of the horses the reason that it
41:14the problem or the mutation
41:24figure two i said yeah
41:27nope that's figure three
41:31so what they did is they looked at um
41:36is that the best one yeah the other
41:37one's the pcr ripple what they did is
41:44exon 16 and exon 18 because the issue
41:48horses was that they were skipping exon
41:51which was an important component of the
41:54obviously all the components are
41:55important but this one especially
41:58encoded one of the tyrosine kinase
42:01and so if you had a pcr
42:10see if i can do both of these things at
42:12once if you had a pcr
42:14product from messenger rna that had
42:23versus a pcr product from
42:28let me make sure i'm using the right
42:29expression if you had messenger rna that
42:31had all three of these
42:32exons and you converted that to cdna
42:40pcr so that's the reverse transcriptase
42:44not pcc and then you do pcr
42:47this would give you a product that's
42:49pretty large right the product for that
42:52like that big if you had messenger rna
42:55where the exon had been skipped as it
42:57was being skipped in the sabino horses
43:00then you could convert that to cdna do
43:04and the product for that would be quite
43:08so that's what they're showing on this
43:12fortunately i still have my markings
43:13there in the mutant which is um
43:17which is lane three let's see do i still
43:21yes i'll use green here so in the mutant
43:24you have a large amount of the
43:28smaller product which means that you're
43:29getting the exon skipping
43:32issue and you have a small amount of
43:35the full product which means that the
43:37mutation doesn't cause exon skipping
43:40every time it just causes it most of the
43:42time about two to one
43:44is the ratio on the other hand if you
43:47homozygous for the wild type as in lane
43:50all of your dna is converted to
43:54that messenger rna includes exon 17 and
43:57so you have the larger
43:58product when you do real-time pcr and
44:01then the heterozygote
44:04again has both mutant transcripts
44:07and wild-type transcripts and it has
44:10that's opposite of the the totally white
44:14horse and so this is the intermediate
44:16where it works most of the time but
44:18about a third of the time
44:20uh you have the the failed exon skipping
44:23transcript so this is why it revealed
44:25the defect by looking at
44:27reverse transcriptase pcr products you
44:30could see how long the messenger rna was
44:32in these different horses and the
44:35having skipped exon 17
44:38you know that reveals what the defect
44:40was it revealed that it was in fact
44:42skipping the exon if all the pcr
44:44products in the real
44:46reverse transcriptase pcr had been the
44:48same link then their hypothesis would
44:50been thrown out the window
44:54okay other questions you may have
44:58while we i guess we don't have much time
45:07i guess we don't have quite as much
45:09volume on this exam as i
45:10glance over the review sheet there so
45:14maybe it won't be as challenging to you
45:17once you get your ducks in a row
45:20but let me know if you have questions
45:21i'll check my email periodically
45:24through the weekend probably and i can
45:25try and respond just ask before
45:27you know it gets too late prior to the
45:29exam the exam will go live at midnight
45:31on sunday night and run through midnight
45:34so you'll have all day monday to do it
45:38as i said meet for class on monday
45:42and we'll pick up on wednesday sorry i'm
45:44looking i'm not just
45:46fading out i'm looking at my calendar on
45:48the floor there so we'll
45:49we'll get back to business on wednesday
45:53and go from there okay so i guess i'll
45:57close if no one has any questions i
45:59owed you a few minutes from the other
