Why is it that when a frame shift occurs (result of an addition or deletion mutation) that only the gene in which the mutation occurs is affected? why isn’t the entire chromosome frame shifted?
Additionally, how is it that when a chunk of DNA is fired at the nucleus of a cell (e.g, with a ‘gene gun’) it seems to have an uncanny knack for slotting into a spot where it is actually functional? Why does this inserted DNA not become just another lump of non-coding DNA?
Thanks for your help,
-Oli
I will answer the second question first: for most cells the vast majority of exogenous DNA that is put in by any means (gene gun, chemical transfection, electroporation, microinjection) does not integrate at all and therefore never becomes coding DNA. IIRC the rate for the cells I used to work with (a type of human tumor cell) was something like one in 1,000 to one in 10,000 copies of DNA electroporated would integrate, depending on conditions. The reason so many of the integrated copies can express is that they are usually themselves intact genes, they have their own promoters and such and are not dependent on the chromosomal DNA for expression, although the location on the chromosome at which they integrated did have bearing on the level of expression.
As for the first question: Keep in mind that genes along a chromosome are generally discrete coding units, with a lot of empty space in between.
Just because one gene has a mutation doesn’t mean the next one down the line will be affected…it isn’t right next door in molecular terms, and it is most likely transcribed separately.
I hope that helps…my technical explanations never seem as clear on the computer screen as they do in my head…
OK, I understand the first part, that kind of makes sense. (though I still don’t understand exactly *how* exogenous DNA is integrated…)
I still don’t quite get the second part, however. Given that a chromosome consists of one continuous DNA molecule, why isn’t everything, coding or non-coding, ‘downstream’ of the mutation frame-shifted as well?
I hope that makes some kind of sense…
I appreciate your help, btw.
-Oli
Well, I guess technically everything downstream IS frame shifted, but it doesn’t matter because most of it is non-coding and isn’t transcribed.
Entire chromosomes are not transcribed and translated as one unit, the individual genes along the chromosome are transcribed and translated separately. Does that help?
Genes on a chromosome are generally so far apart that it takes a gross mutation, like an inversion, deletion or translocation to affect multiple genes.
As far as how exogenous DNA is integrated, I believe it is done through the cell’s own DNA repair mechanisms. It is an interesting question to ask whether integrating exogenous DNA
has any evolutionary advantage over time, or is it solely a mistake by the cell’s repair mechanism.
Are you studying this subject in school, or are you just interested?
I’m studying Biology at school (Yr 12), but we don’t go into much depth, just the basics of transcription/translation.
As to the evolutionary advantage of integrating exogenous DNA… I read something on a similar note recently regarding mitochondrial DNA.
Apparently, mitochondria retain only a fraction (~5%?) of the genetic material they had befor they entered into symbiosis with eukaryotic cells. A large proportion of the original mDNA has ended up located as pseudogenes in the genome of the ‘host’ cell. When the mitochondria lyse, their DNA ends up floating around the cytoplasm, and some of it ends up being integrated into the genome.
Slightly off track, but I thought it might be of interest.
So is it the same deal with retroviruses, as far as integration being totally random? That is, when a retrovirus (ie HIV) reverse transcribes it’s genetic material, is it purely luck that it ends up in the right spot in the genome?
And if so, why do no virally infected cells become cancerous? If you are shoving large chunks of DNA into random positions in the genme, surely it stands to reason that at least some cells infected will become cancerous?
-Oli
I think the fact that Laughing Lagomorph has neglected to explicitly point out, and which may be causing your confusion, concerns stop codons. When there is a frameshift mutation, somewhere downstream there will randomly occur a stop codon – one of three codons which tell the transcription machinery to shut down. This may occur within the mutated gene, in the “junk” after the gene, or in a gene further down the line (so more than one gene may actually be affected by this type of mutation), but a stop codon will eventually occur. After that, there may be a section of unreadable garbage (if the stop codon occurred in the middle of some important region), but eventually there will be another start codon, and transcription will be able to start back up in the proper frame. Oh, also I should point out that the entire chromosome is not in the same frame – hell, whole genes are not necessarilly in the same frame. Introns and intergenic regions may be of any length (putting adjacent regions in any frame). All that is necessary is that there are clear “roadsigns” that tell where transcription starts and stops. Only what is between those signs needs to be in the correct frame.
As for retroviruses, I strongly suspect that they have “machinery” (DNA cutting and splicing proteins) to insert their genetic code into host cells, and do not rely on being spontaneously absorbed. Also, some viruses do cause cancer.
Ah. That clears things up a bit. I know some viruses cause cancer - is this a function of the mechanism by which they integrate DNA into the host genome, or is it always a secondary effect? (e.g, HIV damaging immune system and making one more vulnerable to cancers)
If retroviruses have DNA cutting and splicing enzymes, how do they transport them to the nucleus?
My understanding was that viral RNA ends up in cytoplasm, devoid of protein shell. DNA mirror of the RNA built. DNA then integrated into genome.
Are the viral enzymes attached like histones are to DNA, or similar?
-Oli
Actually, stop codons have nothing to do with the reason that other genes aren’t affected by the frameshift mutations… sorry. 
Short version of this discussion: transcription doesn’t use codons, so a frameshift doesn’t change anything in transcription (promoters and terminators still look the same to the transcription machinery). Each transcribed gene has its own promoter and terminator (ok, I’m waving my hands a little, and including “polyadenylation signal sequence” in the “terminator” category). There’s junk in between a terminator for one gene and the next promoter anyway, so one more or less bit of junk doesn’t matter.
If the frameshift is before a promoter… no biggie, the promoter still has the same sequence, so the frameshift does nothing. If it’s in the promoter, the gene might not be transcribed (although promoters can be rather variable, so it might still work). If it’s between the promoter and the terminator, it’s a frameshift mutation within that gene.
Now lets get to the other end. If there’s an insertion or deletion before the terminator, the terminator still looks like a terminator, so transcription terminates. If the insertion or deletion is after the terminator, nothing is affected (see the promoter discussion above). If it’s WITHIN the terminator, chances are the terminator will still work… or the polymerase will continue to transcribe the gene until it falls off on its own or hits another terminator.
You are completely correct, of course. Please forgive the trascription/translation brain-fart. What I was mostly describing would be the effect of a frameshift on translation of the RNA transcript into protein. So, to backpedal and correct what I said, if you get an RNA transcript with a frameshift mutation (the transcription of which should work fine, as jharmon pointed out), the translation machinery will start at the start codon just fine, go along and past the frameshift mutation, and continue until it reaches a new stop codon, or the end of the transcript. So the end product will either be a truncated protein, or a protein with a normal first section, and meaningless amino acids in the second section.
(I was going to speculate on whether introns would be correctly excised (my guess is yes), but given my previous screw-up today, I’ll let someone else stick their neck out for that one. ;))
Your guess is correct. Again, introns don’t care about codons–if their sequences remain intact (really, if just certain parts of their sequences remain intact), they’ll be excised. It depends a little on the type of intron, but for the most part you can assume they’d be excised (I believe this is used in some experimental systems–if your tags that allowed you to insert a sequence into a gene are within an intron, they’ll be spliced out, and thus won’t affect anything; if anyone is still interested in a week or so, feel free to top this post and I’ll try to reply then–I’m working on some Flash animations on these topics right now, and am reading a lot of research for ideas for the interactive sections)…
In the case of HIV, integrase targets highly bent segments of the host DNA. These correspond to areas that are being actively transcribed. In my medical biochem class, we saw a map that showed the most common places for HIV integration, which is also a good way to look at the most actively transcribed regions of human DNA. Presumably, since these sites are so active, it increase the chances that the viral DNA sequence will be transcribed.
As for the cancer, I don’t know why, at least in the case of HIV. Perhaps so many cellular resources are being used to make little HIV particles that the cell doesn’t have time to make abnormal cellular products? But I do know that infections of the papilloma virus are linked to cervical cancer in women.
IIRC, insertion of exogenous DNA isn’t always random.
Wasn’t there a French gene-therapy trial were the gene vector introduced to cure the disease inserted into a region of the genome where it later caused leukemia?
Again if IIRC, it was initially trumpeted as an example of the power of gene therapy; the transfected gene alleviated the disease (and I can’t remember the disease/details off the top of my head). But then months/years later, leukemia appears in a large percentage of the patients.
The vector apparently prefered to insert in a regulatory region involving a gene whose disruption would lead to leukemia.
As an aside, what I find interesting is that HIV genes encode for splice variants. If I have this right, HIV produces mRNAs in the nucleus that encode one protein when some exons/introns are not spliced and a different protein when some exons/introns are spliced.
If I do have this right, how do these HIV mRNAs bypass the cellular mechanisms to degrade misspliced mRNAs. The cell has mechanisms to ensure mistranscribed endogenous mRNAs aren’t exported out to the cytoplasm and ribosomes, right?
OK, back from work!
This is true. In fact I have quite a bit of experience doing transfections of plasmid (nonviral) DNA that was designed to integerate at particular places in the chromosome (granted, at a low rate).
IIRC viruses tend to integrate at prefered locations as well.
bryanmcc I actually started composing a post dealing with stop codons as my original reply to starman’s question, then realized it wasn’t relevant! So don’t feel bad.
starman it is a fascinating field, isn’t it?
This is a really informative thread… thanks all for your patience with my no doubt ignorant Qs!
I feel silly asking this, but how exactly are introns ‘signposted’ for want of a better word?
This is how I understand it: You’ve got a start codon, which signals the start of the exon. A stop codon signals the end of the exon. All the bits in between start and stop codons are introns. Correct?
On another (related) note, does the DNA in introns serve any known function? I know it doesn’t code for protein, but does it serve any other role? (perhaps in a regulatory capacity?)
Thanks again,
-Oli
Don’t feel silly. A lot of this stuff is only now becoming understood by people who do this for a living, so it’s no wonder that a student isn’t exactly clear on how it works. To be honest, if you’d asked this 2 weeks ago I’d have a much harder time answering–I’ve been reading a LOT writing these animations (this most recent batch has been on Homologous Recombination, Site-Specific Recombination, Transposition, Transcription, and Intron Splicing, so you caught me at the right time).
Ok, let’s start with what a eukaryotic pre-mRNA transcript “looks like”. The easiest way to imagine it is a Harry Potter-style scarf (although the spacing isn’t exactly right, but it’s close enough). The burgundy bands are introns, and the yellow bands are exons.
The exons contain everything that will be in the final mRNA. This includes regulatory regions at both the “front” (5’ end) and “back” (3’ end), and of course a start codon, a bunch of other codons to code the protein, and then a stop codon. So the final post-processing mRNA looks vaguely like this:
5’ cap; regulatory sequences (promoters, activator binding sites, etc); start codon; the rest of the protein; stop codon; more regulatory sequences (there can still be activator and repressor binding sites down here–remember, RNA is flexible, so this part can curve around and shut down or instigate translation); a poly(A) tail.
Now to do what I can to answer your questions. Unfortunately, I’ve only just begun my intron review, and that was on the group I introns, which are somewhat atypical. Hopefully someone can either say, “Yep, that’s right” or “Yeah, well, not so much”
AFAIK, introns tend to be flanked by sequences that basically say “hey, cut me out!” to the cell. In other words, they’re signposted with sequences that the spliceosome can recognise and bind to.
As for their purpose… I don’t think we’re 100% certain, but as soon as you start looking at things like the alternative splicing that was already mentioned, you can see at least one way in which they’re incredibly useful. Basically, they allow different tissues to express different proteins from the same gene (by splicing out different introns). On top of that, they allow genes to “shuffle” without things being TOTALLY random–if an exon moves to another gene (bringing along enough of the introns around it to be likely to get spliced, and moving through one of the various recombination processes), it’s less likely to screw things up than if you just randomly insert sequences (it’s kinda sorta correct to think of exons as domains on the final protein, although it doesn’t always work that way… so, if you move the exon(s) that code for a fatty-acid binding domain, it might still do something in it’s new location; in reality, I think an exon is more likely to code for part of a fatty-acid binding domain, but you get the idea).
All that said, I don’t think we really “get it” yet when it comes to the possible uses of splicing. I envision a day when we have several possible protein domains completely understood structurally and enzymatically, and can simple shuffle them around (using exons flanked by intron “splice me” bits) to make proteins with new functions (okay, I need a fatty acid… ribose… transport channel… there, those pieces should do it)…
The only cancer I can think of off the top of my head that’s associated with AIDS is Kaposi’s sarcoma, and that’s caused by Human Herpesvirus 8, not HIV. The association is due to the decreased immune response in AIDS patients, so they’re not able to fight off HHV8 like normal people do.
IIRC, viruses can cause cancer in a couple of different ways. Some do, indeed, cause it by inserting in the right (or wrong, depending on your viewpoint) spot in your DNA. For example, it might separate a gene that promotes cell division from the regulatory sequence that keeps that gene turned off, causing inappropriate cell proliferation.
Other viruses carry genes in their own genome that cause cancer. Often, these are very similar to human cancer-causing genes (like the growth gene in the last paragraph). Presumably, sometime way back when, a virus inserted itself into someone’s genome, and when it popped itself back out, accidentally carried that human gene along with it, and now it’s common among the virus population.
Hey jharmon:
Does it ever work this way? I have never heard of exons corresponding to domains on the final protein. Just wondering.
Also, starman, for an extremely good read, I suggest you check out The Selfish Gene by Richard Dawkins. It may give you a new perspective on how to think about genes. (After that one, read The Extended Phenotype and The Blind Watchmaker- Dawkins is a fantastic writer and a staggeringly brilliant guy.)
If you ask me, for the most part, the answer is “no.” Introns are merely segments of “selfish DNA” that have succeeded in piggy-backing within a functional gene. Again- read Dawkins.
-Apoptosis
I have read Dawkins … “The Selfish Gene” and “Climbing Mount Improbable” - great reads, the both of them. I don’t know if it is quite so simple as introns just being bits of selfish DNA though. I mean, is an intron just a homogenous lump of random, non-coding DNA, or are there different types of intron DNA? Do introns have any discernable order to them as other non-coding regions (eg pseudogenes) do?
-Oli
Hey starman:
As another poster implied above, introns are grouped together based on the manner in which they are excised (i.e. Type I, Type II). So the answer is “yes”: introns have a discernable order. However, you seem to suggest that selfish DNA is “just a homogenous lump of random, non-coding DNA.” This is not necessarily the case. Insertion sequences are examples of selfish DNA, and they are far from random.
While thinking about this, I recalled something that may contradict my suggestion that introns are selfish DNA. Prokaryotes do not possess introns, and simple eukaryotes (like yeast) have very few introns. I do not know how the “experts” explain this.
-Apoptosis