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g3n3h4x0r
Fungal Geneticist

Registered: 09/29/13
Posts: 73
Last seen: 8 years, 9 months
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Cloning, Spore Prints, and Genetics
#18908444 - 09/29/13 06:59 PM (10 years, 3 months ago) |
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Hey everyone, new here! I'm a fairly new grower and everything is going pretty smoothly. I'm also a geneticist working on my PhD, so I love to ask questions. I've been doing more research and found a wide variety of 'mutants,' which got me thinking.
You can view some of these mutants > here < if you had any interest (thanks to user Tmethyl)
Anyway, people usually look at a nice mushroom in the batch and choose that to either clone or create spore prints. I understand fungi as an organism, but not so much the reproductive cycle in relation to cultivation. These mutants made me curious if it were possible to produce more in the case I get one. I would assume the spores are part of sexual reproduction and go through natural DNA scrambling, so you wouldn't expect any (significantly) more mutants if you took a spore print.
What about cloning / creating liquid culture from the mutant?
I'm assuming we got an actual genetic mutant and not just a random environmental fluke during growth. I've done some searching around the forums with little success, so I thought I'd give the question a try.
Why is this important? It probably isn't - just curiosity - maybe catalog some stuff for more knowledge on the site. In the rare case that a mutant grows better than the rest, it could be a future liquid culture to share!
Thanks everyone!
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azur
God of Fuck


Registered: 04/21/12
Posts: 28,103
Loc: Daid
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Re: Cloning, Spore Prints, and Genetics [Re: g3n3h4x0r]
#18908490 - 09/29/13 07:07 PM (10 years, 3 months ago) |
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I've thought your same thought for sometime now. I used to get a decent amount of mutants, so I decided to try it on my next one. And now I don't get mutants. ??
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g3n3h4x0r
Fungal Geneticist

Registered: 09/29/13
Posts: 73
Last seen: 8 years, 9 months
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Re: Cloning, Spore Prints, and Genetics [Re: azur]
#18908676 - 09/29/13 07:50 PM (10 years, 3 months ago) |
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What did you try? Did you try the cloning procedure on the mutants?
Perhaps I need to find a book on the specifics of fungi. It just seems odd that millions of spores are used to colonize a cake and a mushroom would have a single set of genetics, especially considering the nature of the organism. Perhaps it wouldn't ever be possible to isolate a mutant and fungi are simply evolutionary superior in terms of genetic mutation and heredity.
I'm going to find a book, hopefully online. If anyone has any suggestions while looking here, that'd be awesome. Otherwise, I'm sure I can find something in this massive forum. =]
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Amphibolos
Le bourgeois gentilhomme




Registered: 05/22/09
Posts: 626
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Re: Cloning, Spore Prints, and Genetics [Re: g3n3h4x0r]
#18908790 - 09/29/13 08:22 PM (10 years, 3 months ago) |
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I like the book from Carlile, called the fungi
Here is a link from my dropbox.
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"Homo sum ; humani nihil a me alienum puto"
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azur
God of Fuck


Registered: 04/21/12
Posts: 28,103
Loc: Daid
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Re: Cloning, Spore Prints, and Genetics [Re: Amphibolos]
#18908870 - 09/29/13 08:39 PM (10 years, 3 months ago) |
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I didn't try anything because I haven't see a mutant since I wanted to.
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Cloning, Spore Prints, and Genetics [Re: azur]
#18913147 - 09/30/13 08:54 PM (10 years, 3 months ago) |
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The mutants are just developmental deformities for the most part. I assume you're talking about abhorts.
True mutants have produced plenty of interesting strains like PE and the red spores.
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It just seems odd that millions of spores are used to colonize a cake and a mushroom would have a single set of genetics, especially considering the nature of the organism.
There's lots of disinformation or just plain old ignorance here. Plenty from the "experts" here too.
Mushroom genetics are just like the genetics of any sexually reproducing organisms. And there's certainly more variation in the fungi kingdom than in plants or animals.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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Gretchenmeister
Starbeing/Psilocybin Savant



Registered: 07/23/05
Posts: 1,032
Loc: From the Stars
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Re: Cloning, Spore Prints, and Genetics [Re: fastfred]
#18913584 - 09/30/13 10:21 PM (10 years, 3 months ago) |
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fastfred said: The mutants are just developmental deformities for the most part. I assume you're talking about abhorts.
True mutants have produced plenty of interesting strains like PE and the red spores.
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It just seems odd that millions of spores are used to colonize a cake and a mushroom would have a single set of genetics, especially considering the nature of the organism.
There's lots of disinformation or just plain old ignorance here. Plenty from the "experts" here too.
Mushroom genetics are just like the genetics of any sexually reproducing organisms. And there's certainly more variation in the fungi kingdom than in plants or animals.
-FF
I fucking love FastFred... I am curious why he does not have a TC tag yet.....
FastFreds Media Cookbook is the raddest collection of mushroom media recipes on the planet. Thanks for all your contributions FF, I have learned tons from you over the years. Your information and advice is always well rooted in proven science and fact.
-------------------- What's wrong with folks? Point your IRC client to irc.socialirc.com, port 6667, #cultivation and #shroomery for live chat with like minded hobbyists and connoisseurs. Mush Porn
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g3n3h4x0r
Fungal Geneticist

Registered: 09/29/13
Posts: 73
Last seen: 8 years, 9 months
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Re: Cloning, Spore Prints, and Genetics [Re: fastfred]
#18915932 - 10/01/13 12:22 PM (10 years, 3 months ago) |
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Appreciate the input! Also thanks to Amphibolos for my recent reading.
First of all, I was extremely vague, apologizes.
Second, I don't think I made a big deal of the spore concept, but I'll clarify.
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It just seems odd that millions of spores are used to colonize a cake and a mushroom would have a single set of genetics, especially considering the nature of the organism.
So basically, haploid spores are responsible for the initial creation of the also haploid mycelium. These mycelium combine to form diploid mycelium, which turns into the fruiting body, which we know and love.
Now, we inject a large number of spores into a cake to colonize of which a few turn to primary mycelium and grow. Given that there's such a high quantity of the haploid mycelium, if we used spores from a mutant created from a change in a single gene, statistically it's not feasible to expect multiple mutants in a cake created from the spores since they turn to a diploid organism - sexual reproduction.
Here is the question I can't get answered from a text, however. If I were to identify a genetic mutant, could I create liquid culture from a diploid fruiting body such that a new cake created from this culture would have the same genetics as the mutant?
If this were possible, I would be able to do complementation assays down the road.
On a side note, I have a genetics lab of my own I built and actively do transformation of certain plants through vectors and Agrobacterium. I can easily extend this to fungi. With this equipment, I can genetically identify mutants, so this IS feasible to me. Additionally, I have mutagens functional with fungi such that I could force mutants - but not much text exists to outline protocol, etc.
Thanks everyone!
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deadmandave
Slime


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Re: Cloning, Spore Prints, and Genetics [Re: g3n3h4x0r]
#18919067 - 10/01/13 11:45 PM (10 years, 3 months ago) |
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It is my understanding that in a multi spore grow, one mushroom can contain a multitude of genetics. whether it is the combination of these genetic lines that create a mutant or one dominant diploid is not clear to me.
However if you did clone a mutant onto a petri dish, you should be able to see sectoring from different strains and from there you could test grow each one; or use the equipment you have which sound incredibly fancy and expensive
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RogerRabbit
Bans for Pleasure



Registered: 03/26/03
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Re: Cloning, Spore Prints, and Genetics [Re: deadmandave]
#18919769 - 10/02/13 07:02 AM (10 years, 3 months ago) |
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Quote:
What about cloning / creating liquid culture from the mutant?
Cloning a mutant will result in more flushes with mutants. I've done this many times. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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g3n3h4x0r
Fungal Geneticist

Registered: 09/29/13
Posts: 73
Last seen: 8 years, 9 months
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Re: Cloning, Spore Prints, and Genetics [Re: RogerRabbit]
#18920349 - 10/02/13 10:36 AM (10 years, 3 months ago) |
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Quote:
It is my understanding that in a multi spore grow, one mushroom can contain a multitude of genetics. whether it is the combination of these genetic lines that create a mutant or one dominant diploid is not clear to me.
Ah! This is what I was looking for, but not what I was hoping for. Thanks!
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However if you did clone a mutant onto a petri dish, you should be able to see sectoring from different strains and from there you could test grow each one; or use the equipment you have which sound incredibly fancy and expensive
I did hear much about this now, and I think this is the direction I'll go. As for the equipment, it was not too expensive (nothing over $500, at least). I'm a big part of an open source movement with people, so I try to build the equipment with people rather than buying from a company. Can save over 10 fold on most things and know how they work, so you can actually fix them.
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Cloning a mutant will result in more flushes with mutants. I've done this many times. RR
Excellent news! Thank you!
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Cloning, Spore Prints, and Genetics [Re: g3n3h4x0r]
#18921122 - 10/02/13 01:15 PM (10 years, 3 months ago) |
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g3n3h4x0r said: So basically, haploid spores are responsible for the initial creation of the also haploid mycelium. These mycelium combine to form diploid mycelium, which turns into the fruiting body, which we know and love.
Now, we inject a large number of spores into a cake to colonize of which a few turn to primary mycelium and grow. Given that there's such a high quantity of the haploid mycelium, if we used spores from a mutant created from a change in a single gene, statistically it's not feasible to expect multiple mutants in a cake created from the spores since they turn to a diploid organism - sexual reproduction.
The monokaryotic myc coming from a spore grows pretty slow and weak. With millions of spores that are often even clumped the myc spends almost no time in the monokaryotic state.
Once mated the myc is dikaryotic, which is the normal state of the organism. During spore production you get nuclear fusion of the two nuclei, then two mitotic events. So you end up with 4 spores accounting for 100% of the parental genetics. You can see with a scope that the spores form in tetrads.
I think this is where people have gotten the idea the the parental genetics are recovered every generation. It's very likely that these 4 spores will land in a clump, germinate, and mate with each other, recovering the parental genetics.
Workman came up with the easiest mating method I've seen yet. He grows up a jar/culture of monokaryotic myc then adds spores of the desired parent. Odds are that these germinating spores will be very close to the already grown monokaryotic myc and mate with it.
Ways to get good spore solutions with unclumped spores include shaking, ultrasonic treatment, and surfactants like Tween or JetDry (dishwasher rinse agent).
Here's a great pic to help explain the mating mechanism...


Here is a good explanation of the lifecycle basics...
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In general, the life cycle involves the fusion of hyphae from two individuals, forming a mycelium that contains haploid nuclei of both individuals. The fusion of hyphae is called plasmogamy. The fused hyphae containing haploid nuclei from two individuals is heterokaryotic. In some cases, plasmogamy results in cells with one nucleus from each individual. This condition is called dikaryotic. Eventually, two nuclei that originated from different individuals fuse to form a diploid zygote. Meiosis then produces either four haploid nuclei or four haploid cells.


All this can be hard to keep straight, so the last pic is very helpful.
Mating experiments are fun to do, I suggest you try a few. Just prepare a spore solution using some surfactant and some manner of agitation. Dilute the spore solution down and dial in your micropipettor so that you're getting 0-2 spores per drop (obviously 1 is the target). Then spot away on petri dishes. You can check your results with any old scope you have around and mark the spots you got 1 spore. You can transfer the spores to different dishes or tubes, or let them grow out a bit before you transfer them.
Now you should have a good selection of monokaryotic cultures, and you can transfer any two of them to a single dish and watch the results when they meet. They'll usually either form a line of inhibition, grow right past each other like they don't even notice each other, or form a ridge of dense growth that will sector out.
To confirm mating you need to check for clamp connections. You'll need a 1000X oil lense IIRC. Here's a pic I took...
 Just to the right of center is the clamp connection. This is proof positive of a dikaryotic/heterokaryotic culture.
At some point I'd love to try mating some related but normally incompatible cultures together. We've had lots of discussions here on that. Everything from protoplast fusion to rattlesnake venom has come up. RR has actually done some rattlesnake venom work.
One thing that would be really useful would be to find some auxotrophic mutants. That would allow some really cool mating work to be done. The utility of auxotrophs is that you can devise a complementary auxotroph mating scheme and grow on minimal media and you're assured that any growth is from the desired cross.
I'm curious what sort of mutation scheme you have in mind? UV is pretty easy, and I've thought of trying some formaldehyde cross-linking. It would be nice to come up with some more interesting mutations.
Agro. works on fungi. Plenty of papers out there on it. Fungicide resistance and GFP have been talked about before here. If you have some of those vectors on hand it would be a fun thing to try. Finding an effective promoter might not be the easiest thing in the world, but then again you might get lucky or maybe the pol isn't all that picky.
If you want the most obvious GE trick... throw a few copies of TDC into them. 
GFP has also been talked about quite a bit, and I've seen a few papers that got good GFP expression in fungi. IIRC most of them found that the presence of an intron was key to getting good expression. So that might be something to consider. Seems kind of odd to me that genomes that small would require introns in everything, but who knows?
I'd start with some simple mating experiments, and get your mating types worked out. It's easy to type any monokaryotic culture if you have the 4 types already isolated. Just grow it out dime sized or use a larger wedge of your sample, then transfer your 4 typing cultures around it. Aside from typing the culture this is an easy way to do crosses also, just grab a wedge of a sector from the quadrant that mated.
You can find my media cookbook here... http://www.shroomery.org/9393/FastFreds-Media-Cookbook
If you find any good recipes, let me know! There's plenty of work that still needs to be done on nailing down a good mushroom minimal media and determining the nutritional requirements of our species of interest.
-FF
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g3n3h4x0r
Fungal Geneticist

Registered: 09/29/13
Posts: 73
Last seen: 8 years, 9 months
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Re: Cloning, Spore Prints, and Genetics [Re: fastfred]
#18921941 - 10/02/13 03:32 PM (10 years, 3 months ago) |
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Boom! You're awesome! I was going to give you a rating, but I can't do that yet. I'll remember when I hit that 50 post mark. 
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The monokaryotic myc coming from a spore grows pretty slow and weak. With millions of spores that are often even clumped the myc spends almost no time in the monokaryotic state.
This is the kind of information that's painstaking to find. I was about to resort to reading journals, which who knows how much worse it would be finding a very good scholarly article on an illegal substance. I've had my troubles with my work on marijuana. This is the trouble with text books, everything happens according to the text, but you never know which it prefers, how long it's in a stage, etc.
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IIRC most of them found that the presence of an intron was key to getting good expression. So that might be something to consider. Seems kind of odd to me that genomes that small would require introns in everything, but who knows?
This is surprisingly true and something I always make sure to do when creating vectors. Eukaryotic systems of all kinds prefer to remove an intron from your expressed gene. In part, this is due to the transcript recruiting the splicing machinery anyway and the need for it to run due to timing and chemical/energy reasons - timing is one thing I've always found extremely odd, especially considering evolution. I can explain more later if anything, my recent ventures have got me behind with classes - I'll write up a paper and put some stuff together when I get to the real work and get caught up!
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Agro. works on fungi.
I'll probably be using it. I do some work with various plants and I've been toying with algae. Might trash the algae project to devote more resources to the fungi - they're so much more interesting.
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You can find my media cookbook here... http://www.shroomery.org/9393/FastFreds-Media-Cookbook
Awesome! I was actually looking for some of these and getting really pissed with search engines. Although, I'm not able to browse it?
Seriously, thanks a ton! I'll send you any information I get. I'm still in my elementary stages of just growing the mushrooms, so I have a bit left to learn before I really get to work. I just like to stay way ahead of the game.
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Quote:
g3n3h4x0r said: Eukaryotic systems of all kinds prefer to remove an intron from your expressed gene. In part, this is due to the transcript recruiting the splicing machinery anyway and the need for it to run due to timing and chemical/energy reasons
Processed, mature mRNA gets translated. No sense trying to translate unprocessed mRNAs.
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Gretchenmeister said: I fucking love FastFred... I am curious why he does not have a TC tag yet.....
FastFreds Media Cookbook is the raddest collection of mushroom media recipes on the planet. Thanks for all your contributions FF, I have learned tons from you over the years. Your information and advice is always well rooted in proven science and fact.
Thanks!
If I ever become a "trusted cultivator" from parroting the general basics and blindly agreeing with whoever seems to be in charge... I'd ask for it to be removed.
No diss to TCs and the idea of yet another rating system. I've been around a lot longer than any of the rating systems, and they're nice, but cause as much trouble as they're worth.
-FF
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g3n3h4x0r
Fungal Geneticist

Registered: 09/29/13
Posts: 73
Last seen: 8 years, 9 months
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Re: Cloning, Spore Prints, and Genetics [Re: fastfred]
#18931209 - 10/04/13 01:21 PM (10 years, 3 months ago) |
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Processed, mature mRNA gets translated. No sense trying to translate unprocessed mRNAs.
I'm talking here about transcription. The transcriptional machinery, including those that splice, have higher fidelity on genes with an intron in actually creating mRNA to splice. The existence of an intron would have no effect on an mRNA as it is usually spliced at the same point of transcription. The machinery likes the intron because it sort of 'satisfies' the splicing machinery and keeps it from moving too quickly along the transcript. A sort of 'racing' mechanism is responsible for detachment of the transcript, so without the existence of an intron, the splicing stuff slides along too quickly causing issues.
Simply put, higher fidelity during transcription leads to more mRNA product to be properly translated down the road.
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Cloning, Spore Prints, and Genetics [Re: g3n3h4x0r]
#18941621 - 10/06/13 06:19 PM (10 years, 3 months ago) |
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> The existence of an intron would have no effect on an mRNA as it is usually spliced at the same point of transcription.
You're getting into semantics. Weather transcription and processing are coupled, instantaneous, or done later doesn't matter.
I've never heard of splicing affecting fidelity. That would be news to me, so I'd love to hear more.
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A sort of 'racing' mechanism is responsible for detachment of the transcript, so without the existence of an intron, the splicing stuff slides along too quickly causing issues.
Splicing isn't done by the polymerase. When I took eu. gen. it was taught as separate steps, but mentioned that it often occurs prior to release from the pol..
I've always understood that the reason spliced mRNAs are sometimes translated more efficiently is because processed mRNA has attached proteins that remain after the splicing. They are marked as processed in this manner, which protects them from degradation and/or helps them get translated in some manner.
I'd have to dig a bit, but I can probably come up with something if you're interested. But it makes sense. There's no point in translating unprocessed mRNA, so there must be a mechanism to prefer processed mRNA and let unprocessed mRNA have time to be processed or degrade.
Not all eukaryotic genes have introns, and adding an intron doesn't always seem to help. I have no idea about the specifics of what mechanisms are involved with those effects. Seems to me that it's probably safer to add an intron since I've never heard of it hurting anything.
-FF
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g3n3h4x0r
Fungal Geneticist

Registered: 09/29/13
Posts: 73
Last seen: 8 years, 9 months
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Re: Cloning, Spore Prints, and Genetics [Re: fastfred]
#18957382 - 10/09/13 11:49 PM (10 years, 3 months ago) |
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The existence of an intron would have no effect on an mRNA as it is usually spliced at the same point of transcription.
This is true.
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There's no point in translating unprocessed mRNA, so there must be a mechanism to prefer processed mRNA and let unprocessed mRNA have time to be processed or degrade.
This is true, and the cell doesn't do this.
The main thing here is we're not talking about about transcription vs. not transcription; we're talking about 30% transcription vs. 70% transcription (ex). The introduced gene gets expressed either way. The particularly interesting thing is when an intron is introduced, for some reason the gene product increases (sometimes).
Now, it's safe to say this does not occur during the translation step, as it's the difference of an intron in the DNA.
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I've always understood that the reason spliced mRNAs are sometimes translated more efficiently is because processed mRNA has attached proteins that remain after the splicing. They are marked as processed in this manner, which protects them from degradation and/or helps them get translated in some manner.
This is a sketchy area where we don't have much information, or can get much information (at the moment at least, especially in vivo). Fidelity is a more broad term, where if x polymerase and y polymerase are this productive, and z protein increases fidelity by an arbitrary number, then you get a grand total of xyz fidelity in creating errors (ie. 1/10,000 in creating an error). Given the spliced loops, identified sequences, etc., it's not definite where this occurs. Even if it is proteins added, it would occur during a splicing process increasing fidelity (susceptible to redefinition).
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Splicing isn't done by the polymerase. When I took eu. gen. it was taught as separate steps, but mentioned that it often occurs prior to release from the pol..
Splicing is not done by the polymerase. It's a competition between the polymerase and the RNAs that perform the splicing. Basically, if the splicing mechanism catches up with the polymerase, it can prematurely detatch the transcript in error. There is temperature-based evidence for this - I can get that if I can find a hard drive full of papers (recently moved states). If the introduced gene has some funky gene pattern in it that can speed up the splicing mechanism (by G,C's low chemical bond), it can catch up. This is still a hypothesis, and I would imagine it would vary from gene to gene.
Basically, no one is right or wrong here. I was more-so stating the more popular hypothesis to why a newly introduced gene functions better with an intron - although both are technically correct given current data.
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I'd have to dig a bit, but I can probably come up with something if you're interested
If you have anything, I'd love to read it! I sift through tons of papers, but they tend to be too detailed in something I'm disinterested in. I love this talk!
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Cloning, Spore Prints, and Genetics [Re: g3n3h4x0r]
#18957985 - 10/10/13 05:13 AM (10 years, 3 months ago) |
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Quote:
g3n3h4x0r said: Now, it's safe to say this does not occur during the translation step, as it's the difference of an intron in the DNA.
It can't happen during transcription. Pol doesn't care what it's transcribing.
I guess splicing proteins can attract the polyadenylation machinery in some cases, but then we're just back to talking about processing prior to termination. My point is that it's already been transcribed weather or not it's still attached to pol.
Spliced/processed mRNA will have splicing proteins like SR still attached. That can affect export and translation. Many things will affect ribosome affinity.
See... http://genomebiology.com/2009/10/10/242/figure/F6?highres=y
SR will attach to specific sites in intronless mRNA, or remains attached after participating in splicing. There's also other proteins involved that can protect and help export mature mRNA.
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It's a competition between the polymerase and the RNAs that perform the splicing. Basically, if the splicing mechanism catches up with the polymerase, it can prematurely detatch the transcript in error.
I think you're getting this mixed up with something else. Splicing is mainly done by proteins (99%), ribozymes don't account for that much.
Actually, I take that back... I guess they've taken to calling them "small nuclear ribonucleoproteins (snRNPs / snurps)".
Splicing proteins don't run with the RNA, they attach to specific splice binding sites. They can't attach until the RNA has been transcribed.
Mol. Bio. is just so damn interesting and complex with all it's little tricks! So much so that small-minded folks assume it must be the work of some magic ghost in the sky!
Every little trick is so simple that it actually seems crafty. To me the fun part is understanding things enough that they don't seem like impossible magic. Just millions of simple tricks.
Regulation happens at all levels. The more we learn the more regulation methods we find.
Getting desired expression is like trying to "hack" a mechanical watch.
-FF
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g3n3h4x0r
Fungal Geneticist

Registered: 09/29/13
Posts: 73
Last seen: 8 years, 9 months
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Re: Cloning, Spore Prints, and Genetics [Re: fastfred]
#18961166 - 10/10/13 07:43 PM (10 years, 3 months ago) |
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I'll get diagrams later. You're mixing up a lot of what I saying, unless I'm not being clear. I'm discussing the actual protein interactions that comprise the transcriptome and spliceosome in order to get the job done. There are many coupling interactions that confuse the transcriptional process even more, or else we would know by now why an intron, in some cases, increase overall yield.
I don't think we're on the same topic here, which is why there's confusion.
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fastfred
Old Hand



Registered: 05/17/04
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Loc: Dark side of the moon
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Re: Cloning, Spore Prints, and Genetics [Re: g3n3h4x0r]
#18963794 - 10/11/13 12:36 PM (10 years, 3 months ago) |
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All sorts of things affect transcription frequency. I've never heard of the sequence of the reading frame affecting this though.
I guess certain sequences could stall the pol, or protein binding to certain sequences could stall things. But that is rare, and would only happen to some rare, odd sequences.
I've never heard of any normal regulation mechanism being located in the ORF. Maybe the rare gene that has a downstream enhancer in the ORF, but nothing common.
I just don't think that anything in the ORF is normally going to affect transcription rate. The regulation must be post-transcriptional.
There's plenty of post-transcriptional regulation mechanisms, so I just don't see any reason to think that an intron in the ORF would have any effect on transcription.
-FF
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